Antigen-binding agents that specifically bind epidermal growth factor receptor variant iii

ABSTRACT

The present disclosure relates to antigen-binding agents that specifically bind to epidermal growth factor receptor variant III (EGFRvIII). Antigen-binding agents of the present disclosure include antibodies and antigen-binding fragments thereof, chimeric antigen receptors (CARs) and bi-specific T-cell engagers (BITE!), bispecific killer cell engagers (BiKEs) and bispecific killer cell engagers (TriKEs). Nucleic acid molecules and vectors expressing antibodies, antigen-binding fragments, CARs, BiTEs, BiKEs or TriKEs are also encompassed by the present disclosure. Immune cells engineered to express CARs, BiTEs, BiKEs or TriKEs may be used to specifically recognize and kill cells expressing EGFRvIII.

PRIORITY CLAIM

This patent application is a national stage filing under 35 U.S.C. § 371 of international application No. PCT/CA2020/050378 filed on Mar. 23, 2020, which claimed priority to U.S. provisional application No. 62/824,391 filed on Mar. 27, 2019, the entire contents of which is incorporated herein by reference.

SEQUENCE LISTING

The Sequence Listing submitted as an ASCII text file via EFS-Web, entitled “Replacement_Sequence_Listing_USSN17441706_ST25.txt”, created on Apr. 7, 2022, and of 180 KB, is incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates to antigen-binding agents that specifically bind to epidermal growth factor receptor variant III (EGFRvIII). Antigen-binding agents of the present disclosure include antibodies and antigen-binding fragments thereof, for use in immunotherapeutic modalities including but not limited to chimeric antigen receptors (CARs), bi-specific T-cell engagers (BiTE™), bispecific killer cell engagers (BiKEs) and trispecific killer cell engagers (TriKEs). Nucleic acid molecules and vectors expressing antibodies, antigen-binding fragments, CARs, BiTEs, BiKEs or TriKEs are also encompassed by the present disclosure. Immune cells engineered to express CARs, BiTEs, BiKEs or TriKEs may be used to specifically recognize and kill cells expressing EGFRvIII.

BACKGROUND

EGFRvIII is a well-known tumor-specific mutation of epidermal growth factor receptor (EGFR) that consists of an in-frame deletion of exons 2-7 near the amino terminus of the extracellular domain that removes 267 amino acids from the extracellular domain encompassing the ligand binding domain. This deletion mutant is amplified and highly expressed in 25-64% of high-grade gliomas especially in glioblastoma multiforme (GBM). This genetic alteration has also been detected in a subset of carcinomas of the breast as well as in head and neck squamous-cell carcinoma (HNSCC) using multiple complementary techniques (reviewed in Gan H. K., et al 2013). Numerous studies show that normal tissues are devoid of EGFRvIII, thus making this an ideal cell-surface tumor target amenable to immunotherapy. Clinical evaluation of EGFRvIII targeted immunotherapy including monoclonal antibodies, vaccines and chimeric antigen receptor (CAR)-T cells have/are been conducted with no reports of serious adverse effects; however, none have been approved for therapeutic applications.

CAR-T therapies have revolutionized cancer therapy approaches with phenomenal cure rates (50-90%) in patients with previously incurable aggressive forms of B cell leukemia leading to the regulatory approval of two CD19 targeted CAR-T products for the treatment of B cell malignancies. Despite these high cure rates in liquid tumors, success of CAR therapy in solid tumors have been limited up to date. This is due to a multitude of reasons including the lack of appropriate tumor specific antigens leading to on-target but off-tumor toxicity and tumor mediated barriers that prevent adequate penetration of CAR-modified immune cells and immune suppressive factors in the tumor microenvironment that negatively impact the functionality of CAR modified immune cells. Thus, the development of new CAR therapies to target solid tumors is an area of active investigation. With the tumor specific expression of EGFRvIII which is not present on normal healthy tissues; specific targeting of tumors by immune cells would provide great therapeutic potential with good safety margin for cancers expressing EGFRvIII. Targeting EGFRvIII using novel CAR-T therapies has shown some promise in clinical trials, but none have yet been approved for clinical application. In the present application, monoclonal antibodies that specifically bind to EGFRvIII and not the wild-type EGFR protein have been used to generate chimeric antigen receptor-T (CAR-T) or -NK (CAR-NK) cells to target human cancers that express EGFRvIII including but not limited to GBM, breast, head & neck or oral cancers.

SUMMARY

The present disclosure relates to antigen-binding agents that specifically bind to EGFRvIII and to nucleic acids encoding same.

More particularly, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to EGFRvIII, as well as chimeric antigen receptors (CARs), bispecific T-cell engagers (BiTE™), bispecific killer cell engagers (BiKEs) and trispecific killer cell engagers (TriKEs) that comprise an antigen-binding domain of such antibodies. Antibodies, antigen-binding fragments and CARs are particularly encompassed by the present disclosure.

In accordance with the present disclosure, the antigen-binding domain may comprise complementarity determining regions of an antibody or antigen-binding fragment that specifically binds to EGFRvIII. More particularly, the antigen-binding domain may comprise the heavy chain and light chain variable regions of an antibody or antigen-binding fragment that specifically binds to EGFRvIII. The antigen-binding domain may be in the form of a single chain variable fragment (scFv).

The anti-EGFRvIII antibodies and antigen-binding fragments thereof of the present disclosure may be selected for their lack of binding to wild type EGFR. Moreover, the anti-EGFRvIII antibodies and antigen-binding fragments thereof of the present disclosure may be selected for their lack of internalization in cancer cells. The anti-EGFRvIII antibodies and antigen-binding fragments thereof of the present disclosure may specifically bind to native or denatured EGFRvIII or to an epitope present in both native and denatured EGFRvIII (e.g., linear epitope).

The anti-EGFRvIII antibodies or antigen-binding fragments of the present disclosure may be used as diagnostics and/or therapeutics.

The general structure of the chimeric antigen receptors of the present disclosure is composed of an antigen-binding domain of anti-EGFRvIII antibody, an optional spacer, a transmembrane domain, an optional costimulatory domain and an intracellular signaling domain.

The antigen-binding domain of CARs is generally composed of an antibody's heavy chain and light chain variable regions connected via a linker and forming a single chain (e.g., scFv). The linker may be any linker that allows for the VL and VH chain to form a functional antigen-binding region.

BiTE, BiKE and TriKE molecules may comprise an antigen-binding domain (e.g. scFv) that specifically binds to EGFRvIII and another domain (scFv) that binds to specific immune cells including but not limited to a T-cell specific molecule (e.g., CD3) and NK-cell surface molecules (e.g. CD16). These generally comprise multiple scFvs connected in tandem by flexible linkers.

The antigen-binding domain of CARs or other biologics including and not limited to BiTEs, BiKEs and TriKEs may comprise for example, complementarity determining regions (CDRs) of the antibody heavy chain. The antigen-binding domain may comprise, for example, at least two CDRs of the antibody heavy chain (including for example, at least CDRH3). The antigen-binding domain may more particularly comprise three CDRs of antibody heavy chain. Moreover, the antigen-binding domain may comprise or also comprise CDRs of the antibody light chain. The antigen-binding domain may comprise, for example, at least two CDRs of the antibody light chain. More specifically, the antigen-binding domain may comprise at least three CDRs of the antibody light chain.

A particular aspect of the present disclosure relates to antigen-binding agents (antibodies, CARs, BiTEs, BiKEs, TriKEs and the like) which comprise an antigen-binding domain having three CDRs of the light chain and three CDRs of the heavy chain of the antibody and nucleic acids encoding same. Antibodies, antigen binding fragments, CARs, BiTEs, BiKEs, or TriKEs of the present disclosure may be used in the treatment of cancer.

The present disclosure also relates to cells expressing the antigen-binding agents of the present disclosure and their use in the treatment of cancer.

Exemplary embodiments of antigen-binding agents of the present disclosure include those that bind to a peptide comprising or consisting of amino acid residues 1-76 of human EGFRvIII ectodomain (SEQ ID NO:69), to a peptide comprising or consisting of amino acid residues 3-37 of human EGFRvIII ectodomain (SEQ ID NO:70), and/or to a peptide comprising or consisting of amino acid residues 1-18 of human EGFRvIII ectodomain (SEQ ID NO:71).

More particularly, the present disclosure encompasses antigen-binding agents that bind to a peptide comprising or consisting of amino acid residues 15-37 of human EGFRvIII ectodomain (SEQ ID NO:5).

The antigen-binding agents of the present disclosure may bind to an epitope comprising amino acid residues Cys20 and/or Cys35 in SEQ ID NO:5. More particularly, the anti-EGFRvIII antibody or antigen-binding fragment thereof may bind to an epitope comprising amino acid residues Arg18, Cys20, Gly21 and Cys35 in SEQ ID NO:5.

Particularly, encompassed by the present disclosure, are antigen-binding agents that bind to an epitope comprising amino acid residues Arg18, Cys20, Gly21, Tyr25, Glu26, Glu29, Gly31, Arg33 and Cys35 in SEQ ID NO:5 or to an epitope comprising amino acid residues Val17, Arg18, Cys20, Gly21, Asp23 and Cys35 in SEQ ID NO:5.

The antigen-binding agents of the present disclosure include for example, those that bind EGFRvIII (SEQ ID NO:4) and/or a peptide comprising an EGFRvIII fragment consisting of the amino acid sequence set forth in SEQ ID NO:5 but that are not able to significantly bind a peptide comprising or consisting of the amino acid sequence SCARACGADSYEMEEDGVRKCKK (SEQ ID NO:50) or SCVRACGAASYEMEEDGVRKCKK (SEQ ID NO:54).

The antigen-binding agents of the present disclosure also include for example those that bind EGFRvIII and/or a peptide comprising an EGFRvIII fragment consisting of the amino acid sequence set forth in SEQ ID NO:5 but that are not able to significantly bind a peptide comprising or consisting of the amino acid sequence SCVRACGADSAEMEEDGVRKCKK (SEQ ID NO:56), SCVRACGADSYAMEEDGVRKCKK (SEQ ID NO:57) or SCVRACGADSYEMEEDAVRKCKK (SEQ ID NO:62).

The antigen-binding agents (e.g., antibody or antigen-binding fragment thereof, CARs and BiTEs, BiKEs, TriKEs) of the present disclosure may comprise, for example,

-   -   a CDRL1 comprising or consisting of SEQ ID NO:7, a CDRL2         comprising or consisting of SEQ ID NO:8, a CDRL3 comprising or         consisting of SEQ ID NO:9, a CDRH1 comprising or consisting of         SEQ ID NO:11, a CDRH2 comprising or consisting of SEQ ID NO:12         and a CDRH3 comprising or consisting of SEQ ID NO:13;     -   a CDRL1 comprising or consisting of SEQ ID NO:15, a CDRL2         comprising or consisting of in SEQ ID NO:16, a CDRL3 comprising         or consisting of SEQ ID NO:17, a CDRH1 comprising or consisting         of SEQ ID NO:19, a CDRH2 comprising or consisting of SEQ ID         NO:20 and a CDRH3 comprising or consisting of SEQ ID NO:21, or;     -   a CDRL1 comprising or consisting of SEQ ID NO:23, a CDRL2         comprising or consisting of SEQ ID NO:24, a CDRL3 comprising or         consisting of SEQ ID NO:25, a CDRH1 comprising or consisting of         SEQ ID NO:27, a CDRH2 comprising or consisting of SEQ ID NO:28         and a CDRH3 comprising or consisting of SEQ ID NO:29.

In an additional aspect, the present disclosure relates to a composition or pharmaceutical composition comprising the antigen-binding agents (e.g., antibody or antigen-binding fragment thereof) of the present disclosure and a pharmaceutically acceptable carrier.

Yet another aspect of the present disclosure relates to isolated cells capable of expressing, assembling and/or secreting the antibody or antigen-binding fragment thereof.

The present disclosure also encompasses nucleic acids and vectors encoding and/or expressing antigen-binding agents (e.g., anti-EGFRvIII antibodies or antigen-binding fragments thereof, CARs, BiTEs, BiKEs, TriKEs and the like) of the present disclosure.

Since antibodies or antigen-binding fragments thereof are composed of two distinct chains, the nucleic acid sequence encoding the light chain or light chain variable region and the nucleic acid sequence encoding the heavy chain or heavy chain variable region may be provided on the same or on separate nucleic acid molecules or vectors.

For immunotherapy purposes, nucleic acids encoding CARs or BiTES, BiKEs, TriKEs or purified protein therapeutic forms of these entities are introduced into immune cells of a patient.

The present disclosure therefore particularly relates to nucleic acid molecules encoding or capable of expressing CARs or BiTES, BiKEs, TriKEs. For immunotherapy purposes, BiTES, BiKEs or TriKEs may also be delivered to patients in the form of purified protein.

In a specific and non-limiting example, the isolated nucleic acid molecule of the present disclosure may encode a CAR, BiTE, BiKE or TriKE having an antigen-binding domain comprising CDRs comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13.

In another specific and non-limiting example, the isolated nucleic acid molecule of the present disclosure may encode a CAR, BiTE, BiKE or TriKE having an antigen-binding domain comprising complementarity determining regions comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.

In yet another specific and non-limiting example, the isolated nucleic acid molecule of the present disclosure may encode a CAR, BiTE, BiKE or TriKE having an antigen-binding domain comprising complementarity determining regions comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29.

In accordance with the present disclosure, the antigen-binding domain of the CARs, BiTEs, BiKEs or TriKEs may be in the form of a single chain variable fragment (scFv).

The scFv may have a structure defined by the formula VH-linker-VL or VH-[L]-VL. Alternatively, the scFv may have a structure defined by the formula VL-linker-VH or VL-[L]-VH. The term “VL” refers to the light chain variable region or to a portion thereof, the term “VH” refers to the heavy chain variable region or to a portion thereof and “L” refers to any linker that allows for the linking of the VL and VH to form a single polypeptide chain that allows for the interaction of the VL and VH to form an active antigen-binding region. In a non-limiting example, the linker may be, for example 5-50 amino acids or 10-25 amino acid residues and may comprise any linking sequence known to one of skill in the present art (including longer or shorter linker). An exemplary embodiment of a linker is provided in SEQ ID NO:47.

Exemplary embodiments of scFvs having the formula VH-linker-VL are provided in SEQ ID NOs: 73, 76 and 79. More particular embodiments of scFvs having the formula VH-linker-VL are provided in SEQ ID NOs: 72, 75 and 78. Additional embodiments of scFvs having the formula VH-linker-VL are provided in SEQ ID NOs: 44, 45 and 46.

In order to minimize immune reactions against non-human sequences, antibodies, antigen-binding fragments, CARs, BiTEs, BiKEs or TriKEs may comprise human or humanized framework amino acid sequences.

CARs of the present disclosure therefore comprise an antigen-binding domain of an anti-EGFRvIII antibody, a spacer (i.e., a hinge) and a transmembrane domain for proper anchoring at the cell membrane. CARs may also comprise at least one intracellular signaling domain for activation of particular immune pathways. The chimeric antigen receptor may also comprise at least one costimulatory domain helping in the activation process.

In an exemplary embodiment, the isolated nucleic acid molecule may encode an anti-EGFRvIII antibody or antigen-binding fragment, CAR, BiTE, BiKE or TriKE that may comprise an amino acid sequence at least 80% identical to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:10 and/or an amino acid sequence at least 80% identical to the amino acid sequence of the light chain variable region set forth in SEQ ID NO:6.

The isolated nucleic acid molecule may encode, for example, a CAR that may comprise an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:30, SEQ ID NO: 32 or SEQ ID NO:74. The isolated nucleic acid molecule may comprise, for example, a nucleic acid sequence that encodes an amino acid encoded by the nucleic acid sequence set forth in SEQ ID NO:31.

In another exemplary embodiment, the isolated nucleic acid molecule may encode an anti-EGFRvIII antibody or antigen-binding fragment, CAR, BiTE, BiKE or TriKE that may comprise an amino acid sequence at least 80% identical to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:18 and/or an amino acid sequence at least 80% identical to the amino acid sequence of the light chain variable region set forth in SEQ ID NO:14.

The isolated nucleic acid molecule may encode, for example, a chimeric antigen receptor that may comprise an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:33, SEQ ID NO:34 or as set forth in SEQ ID NO:77.

In yet another exemplary embodiment, the isolated nucleic acid molecule may encode an anti-EGFRvIII antibody or antigen-binding fragment, CAR, BiTE, BiKE or TriKE that may comprise an amino acid sequence at least 80% identical to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:26 and/or an amino acid sequence at least 80% identical to the amino acid sequence of the light chain variable region set forth in SEQ ID NO:22.

The isolated nucleic acid molecule may encode, for example, a CAR that may comprise an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:35, SEQ ID NO:36 or as set forth in SEQ ID NO:80.

Particular aspects of the present disclosure relate to isolated nucleic acid molecules encoding a CAR that may comprise:

-   -   an antigen-binding domain of an antibody that specifically binds         to epidermal growth factor receptor variant III (EGFRvIII),         wherein the antigen-binding domain comprises:         -   a CDRL1 comprising or consisting essentially of the amino             acid sequence set forth in SEQ ID NO:7, a CDRL2 comprising             or consisting essentially of the amino acid sequence set             forth in SEQ ID NO:8, a CDRL3 comprising or consisting             essentially of the amino acid sequence set forth in SEQ ID             NO:9, a CDRH1 comprising or consisting essentially of the             amino acid sequence set forth in SEQ ID NO:11, a CDRH2             comprising or consisting essentially of the amino acid             sequence set forth in SEQ ID NO:12 and a CDRH3 comprising or             consisting essentially of the amino acid sequence set forth             in SEQ ID NO:13;         -   a CDRL1 comprising or consisting essentially of the amino             acid sequence set forth in SEQ ID NO:15, a CDRL2 comprising             or consisting essentially of the amino acid sequence set             forth in SEQ ID NO:16, a CDRL3 comprising or consisting             essentially of the amino acid sequence set forth in SEQ ID             NO:17, a CDRH1 comprising or consisting essentially of the             amino acid sequence set forth in SEQ ID NO:19, a CDRH2             comprising or consisting essentially of the amino acid             sequence set forth in SEQ ID NO:20 and a CDRH3 comprising or             consisting essentially of the amino acid sequence set forth             in SEQ ID NO:21, or;         -   a CDRL1 comprising or consisting essentially of the amino             acid sequence set forth in SEQ ID NO:23, a CDRL2 comprising             or consisting essentially of the amino acid sequence set             forth in SEQ ID NO:24, a CDRL3 comprising or consisting             essentially of the amino acid sequence set forth in SEQ ID             NO:25, a CDRH1 comprising or consisting essentially of the             amino acid sequence set forth in SEQ ID NO:27, a CDRH2             comprising or consisting essentially of the amino acid             sequence set forth in SEQ ID NO:28 and a CDRH3 comprising or             consisting essentially of the amino acid sequence set forth             in SEQ ID NO:29;     -   optionally a spacer;     -   a transmembrane domain;     -   optionally at least one costimulatory domain, and;     -   at least one intracellular signaling domain.

Nucleic acids encoding CARs comprising at least one intracellular signaling domain are particularly contemplated.

The intracellular signaling domain may be, for example and without limitation, from CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma RIIe, DAP10, or DAP12.

In accordance with the present disclosure, the CAR encoded by the nucleic acid molecule of the present disclosure may comprise at least one costimulatory domain.

The costimulatory domain may be, for example and without limitation, from CD28, CD27, 4-1BB, OX40, CD7, B7-1 (CD80), B7-2 (CD86), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LyI08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D or a combination thereof.

In accordance with the present disclosure, the nucleic acid molecule may be operably linked to a promoter allowing expression of the CAR in immune cells. Promoters that are active or specific to T-cells or natural killer (NK) cells are particularly contemplated.

The nucleic acid molecules of the present disclosure may be cloned into a vector. In accordance with the present disclosure the vector may be a viral vector, such as, for example, lentiviral vectors, retroviral vector, adenoviral vectors, adeno-associated viral vectors and the like.

In an additional aspect, the present disclosure relates to an isolated cell transformed with the nucleic acid molecule or with the vector disclosed herein.

In yet an additional aspect, the present disclosure relates to an isolated host cell expressing the anti-EGFRvIII antibody or antigen-binding fragment thereof, the CAR disclosed herein or comprising the nucleic acid molecule, or the vector disclosed herein.

In accordance with the present disclosure, the isolated host cell or isolated cell population comprise immune cells. The immune cells may be for example and without limitation, T-cells, NK cells or combination thereof. The isolated host cell or isolated cell population may be of human origin.

The CAR of the present disclosure is expressed at the surface of the host cells. The CAR of the present disclosure may recognize and bind to EGFRvIII expressed at the surface of cancer cells. The cancer cells targeted by the isolated cell population may comprise solid tumors.

In accordance with the present disclosure, the isolated cell population may comprise T-cells, Natural Killer (NK) cells, cytotoxic T-cells, regulatory T-cells, and combinations thereof.

More particularly, the isolated cell population may comprise T-cells such as CD4+ T-cells, CD8+ T-cells or a combination thereof.

Alternatively, the isolated cell population may comprise NK cells or any immune cell capable of expressing the chimeric antigen receptor.

The isolated cell population of the present disclosure may be engineered to express another (a second) chimeric antigen receptor having affinity for another (a second) antigen of the same target or of a different target.

In another aspect, the present disclosure relates to a pharmaceutical composition that comprises the isolated cell population disclosed herein and a pharmaceutically acceptable carrier or excipient.

In accordance with the present disclosure, the pharmaceutical composition may be used for treating a solid tumor using a population of T-cells and/or NK cells engineered to express the chimeric antigen receptor of the present disclosure.

In yet another aspect, the present disclosure relates to a method of treating a subject having a cancer associated with EGFRvIII expression. The method may comprise, for example, administering the anti-EGFRvIII antibody or antigen-binding fragment thereof of the present disclosure to the subject. Alternatively, the method may comprise administering an isolated cell population engineered to express CARs to the subject. The antibody or isolated cell population may be administered in the form of pharmaceutical compositions.

In an exemplary embodiment, the isolated cell population may be autologous to the subject. In another exemplary embodiment, the isolated cell population may be allogenic (from an allogenic donor) with respect to the receiving subject.

In accordance with the present disclosure, the cancer treated by the method of the present disclosure comprises a solid tumor.

Treatment of gliomas, such as for example glioblastoma multiforme is particularly contemplated.

In another exemplary embodiment, the method of the present disclosure provides treatment of carcinoma, such as for example and without limitation, breast carcinoma, head and neck carcinoma or oral carcinoma.

In a further aspect, the present disclosure relates to a kit comprising at least one antibody or antigen-binding fragment, nucleic acid molecule or vector of the present disclosure. Kits that comprise the pharmaceutical composition or the isolated cell population disclosed herein are also encompassed by the present disclosure. The kit may further comprise written instructions for using said isolated cell population for the treatment of a subject having a neoplasm.

In an exemplary embodiment of the disclosure, kits that are used for producing an antibody or antigen-binding fragment thereof may comprise a first vial containing a nucleic acid or vector encoding a light chain or light chain variable region and a second vial containing a nucleic acid or vector encoding a heavy chain or heavy chain variable region.

Kits comprising nucleic acid molecules encoding CARs of the present disclosure are particularly contemplated.

A further aspect of the present disclosure relates to a method of making the antibody or antigen-binding fragment thereof of the present disclosure. The method may comprise culturing a cell comprising nucleic acids encoding the antibody or antigen-binding fragment so that the antibody or antigen-binding fragment thereof is produced. The method may also involve conjugating the antibody or antigen-binding fragment thereof with a cargo molecule.

Another aspect of the present disclosure relates to a method of manufacturing CAR-expressing cell population. The method may comprise introducing the isolated nucleic acid molecule or the vector disclosed herein into a cell. The isolated nucleic acid molecule may integrate into the genome of the cell.

The nucleic acid molecule or the vector disclosed herein may be introduced into immune cells. The immune cells are then grown ex vivo and transfused to a patient. Upon activation, the transduced immune cells may recognize and kill tumor cells expressing EGFRvIII.

Immune cells of human origin may be used to generate the CAR-expressing cell population of the present disclosure. The immune cells may comprise T-cells, Natural Killer cells, cytotoxic T-cells, regulatory T-cells, and combinations thereof. Particularly contemplated are T-cells (CD4+ T-cells, CD8+ T-cells or a combination thereof) or NK cells.

The CAR-expressing cell population may be isolated and/or substantially purified.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1E: represents histograms obtained using flow cytometry on supernatants of selected hybridomas on U87MG cell lines overexpressing wild type human EGFR (U87WT) or EGFRvIII (U87vIII) as indicated.

FIG. 2: is an alignment between the amino acid sequence of the extracellular domains of wild-type human EGFR and EGFRvIII.

FIG. 3A and 3B: show the results of anti-hEGFRvIII mAbs binding properties to various fragments of the EGFRvIII and WT protein cells displayed on yeast cells (+++ represents 95% +/−5% positive yeast cells which is characterized by positive antibody binding with high affinity; ++ represents 70% +/−20% positive yeast cells which is characterized by positive antibody binding with medium affinity; + represents 30% +/−20% positive yeast cells which is characterized by positive antibody binding with low affinity; (+) represents 5-9% positive yeast cells which is characterized by positive antibody binding with very low affinity; +/− represents less than 5% positive yeast cells which is characterized by ambiguous antibody binding, − represents 0% positive yeast cells which is characterized by no binding; nt=not tested

FIG. 4: Schematic illustrating the synthetic assembly and sequence of the 5B7 antigen-binding domain and mouse CD8 hinge (SEQ ID NO: 30) for insertion in CAR vector with the heavy chain portion (underlined), amino acids corresponding to a restriction site (*), linker (L), the light chain portion (underlined).

FIG. 5A: Schematic illustrating different elements and DNA sequences of the modular CAR vector (SEQ ID NO:37), representing an example of CAR cloning strategy.

FIG. 5B: Schematic illustrating 1D2-CAR-TRIM which contains a shorter scFv portion (SEQ ID NO:78)

FIG. 5C: Graph comparing 1 D2-CAR and 1 D2-CAR-TRIM construct using the CAR-J assay. Briefly, Jurkat cells were electroporated with plasmids expressing either full length or trimmed form of the CAR as illustrated in 5B. CAR-J cells were then mixed with EGFRvIII expressing target cells and examined for activation signature (CD69 expression) via antibody staining and flow cytometric analysis.

FIG. 5D: Schematic illustrating potential bi-specific T-cell engagers and bispecific killer cell engagers.

FIG. 6A and 6B: Human CD8 T cell line Jurkat transiently transfected with the EGFRvIII CAR construct which also expressed green fluorescent protein (GFP) using a 2A self-cleaving peptide based multi-gene expressing system. Expression of GFP was quantified using flow cytometry (A) and the surface expression of CAR was detected by flow cytometry using a fluorescent tagged antibody that recognize murine IgG (Goat Anti-Mouse IgG H&L [Alexa Fluor® 594]; Abcam) (B) (5B7=F265-5B7; 3D12=F269-3D12; 1D2=F271-1D2).

FIG. 7: Schematic illustrating the high-throughput screening method for CAR functionality. (A) Jurkat cells electroporated with various CAR constructs were exposed to varying doses of target cells with (U87-vIII, DKMG) or without (U87-MG) specific target expression. CAR constructs found to have inappropriate properties for advancement are also shown (X1, X2) (B) Tonic signaling/auto-activation of each CAR construct was assessed by examining the level of CD69 expression in CAR-expressing (GFP+) cells without additional stimulation. (C and D) The relative increase in expression of CD69 within CAR cells following stimulation with EGFRvIII expressing target cells is shown.

FIG. 8A: Graph illustrating the in vitro functionality of EGFRvIII CAR-T constructs. Jurkat cells electroporated with various CAR constructs (EGFRvIII 5B7 CAR, EGFRvIII 3D12 CAR, EGFRvIII 1D2 CAR, EGFR WT CAR) were exposed to target cells with (U87vIII) or without (U87, OVCAR3 and A20) EGFRvIII target expression for 24 hours. The level of cell activation was measured by quantifying surface expression of CD69 on Jurkat cells (CD45 positive) by flow cytometry.

FIG. 8B: Graph illustrating screening of CAR functionality with EGFRvIII-specific CAR constructs containing hinge elements of varying amino acid length (screening hinge truncations in EGFRvIII-CAR). Jurkat cells electroporated with various CAR constructs which contained hinge elements ranging from very long, L17-CD8h which contains the entire human CD8 hinge domain and an extended glycine linker, to very short, 1CD8h which contains a single C-terminal amino acid from the human CD8-hinge sequence. Jurkat-cells transiently expressing these various CAR constructs were exposed to target cells with (U87-vIII) or without (U87-MG) specific target expression. Target-induced activation of each CAR construct was assessed by examining the level of CD69 expression in CAR-expressing (GFP+) cells using human CD69-specific antibody staining and flow cytometry.

FIG. 9: Graphs showing the tumor volume (mean±SEM) in athymic nude mice (Jackson Laboratory, Barr Harbor, Me.) injected subcutaneously with 1×10⁶ (FIG. 9A) or 4×10⁶ (FIG. 9B) U87vIII human glioblastoma cells expressing EGFRvIII and administered with 10⁵ (FIG. 9A) or 10⁶ (FIG. 9B) human primary T cells expressing EGFRvIII 1D2 CAR or negative control CD19 CAR on day 5 post tumor cell injection. Tumor size (the length and the width) was measured using a digital vernier caliper. Tumor volume was calculated by using the formula: Tumor volume=(0.4) (ab2), where a=large diameter and b=smaller diameter. The level of CD8 T cell infiltration in tumors from two animals each from groups receiving EGFRvIII targeted CAR-T and showing good tumor control was assessed and compared to CD19 CAR-T negative control (FIG. 9C).

FIG. 10: Schematic illustrating the generation of EGFRvIII targeted NK92-CAR cells for in vitro testing of tumor cytotoxicity (FIG. 10A). Graphs showing data of cytotoxicity of control NK92 cells (NK92-no CAR), NK 92 cells expressing EGFRvIII 3D12 CAR construct (NK92-3D12CAR) or EGFR WT CAR construct towards cells expressing EGFRvIII (U87-vIII) or WT EGFR (U87) as measured by LDH (FIG. 10B) or ⁵¹Cr-release assay (FIG. 10C). Wild-type NK92 cells devoid of a CAR construct was used as negative control (NK92 wt).

FIG. 11: Graphs illustrating tumor growth (left panels) and survival (right panels) of tumor-bearing athymic nude mice administered with NK92 cells expressing EGFRvIII 3D12 CAR construct or devoid of CAR expression (NK92-WT or NK92wt). Athymic nude mice (Jackson Laboratory, Barr Harbor, Me.) were injected subcutaneously with 1×10⁶ U87vIII human glioblastoma cells expressing EGFRvIII. On day 2 post tumor cell injection, mice were given either 5×10⁶ wild-type NK92 cells devoid of CAR or NK92 cells expressing EGFRvIII 3D12 CAR (FIG. 11A) or on day 3 post tumor induction animals were given 1×10⁷ EGFRvIII 3D12 NK-CAR or left untreated (FIG. 11B). Animals were monitored for survival and tumor growth. Tumor size (the length and the width) was measured using a digital vernier caliper. Tumor volume was calculated by using the formula: Tumor volume=(0.4) (ab2), where a=large diameter and b=smaller diameter.

FIG. 12: Graph illustrating repression of target cell growth in CAR transduced T-cell/target cell co-cultures. Human primary peripheral blood derived T cells were transduced with EGFRvIII-specific CAR lentivirus (3D12-CAR-T) or treated similarly in the absence of lentivirus (Mock) and grown for several days in culture. CAR-transduced or non-transduced T cells were then placed in co-culture with EGFRvIII antigen expressing target cells which express nuclear localized mKate2-fluorescent protein (U87vIII). Cells were then examined using live fluorescence microscopy (Incucyte™, Sartorius). Graph depicts the relative target cell growth over 6 days as measured via automated counting of mKate2+cells.

FIG. 13: Graphs illustrating tumor growth (left panels) and survival (right panels) of tumor-bearing NOD/SCID/IL-2Rγ-null (NSG) mice. NSG mice (Jackson Laboratory, Barr Harbor, Me.) were injected subcutaneously with 1×10⁶ U87vIII human glioblastoma cells expressing EGFRvIII. On day 7 post tumor cell injection, mice were given either primary human T cells transduced with EGFRvIII-3D12 CAR or left untreated. Tumour growth was then monitored by caliper measurements (FIG. 13A). Tumor size (the length and the width) was measured using a digital vernier caliper. Tumor volume was calculated by using the formula: Tumor volume=(0.4) (ab2), where a=large diameter and b=smaller diameter. Survival is defined as time to humane endpoint (defined as a tumour volume exceeding 2000 mm³) with and without CAR-T treatment (FIG. 13B).

FIG. 14: Graph illustrating screening of bi-specific T cell engager activity with EGFRvIII-specific constructs containing 3D12-scFV sequence linked to OKT3 human CD3-specific scFV. Supernatant from human embryonic kidney (HEK293) cells transiently expressing 3D12-OKT3 bi-specific engager construct were transferred to wells containing Jurkat cells and varying doses of antigen expressing target cells (U87vIII). Target-induced activation of T cells in the presence or absence of bispecific T-cell engager was measured by examining the level of CD69 expression using human CD69-specific antibody staining and flow cytometry. The fold change in CD69 expression with and without bispecific-T cell engager over varying doses of target cell is shown here.

FIG. 15: Graph illustrating repression of target cell growth in T-cell/target cell co-cultures in the presence of a bi-specific T cell engager constructs containing 4E11-scFV sequence linked to OKT3 human CD3-specific scFV. A control bi-specific T cell engager composed of human—CD19-specific scFv linked to OKT3 is shown for comparison. Supernatants from human embryonic kidney cells transiently expressing 4E11-OKT3 bi-specific T cell engager construct or the CD19-OKT3 bi-specific T cell engager construct were transferred to wells containing primary blood derived T cells and EGFRvIII antigen expressing target cells which express nuclear localized mKate2-fluorescent protein (U87vIII). Cells were then examined using live fluorescence microscopy (Incucyte™, Sartorius, USA). Graph depicts the relative target cell growth over 72 hours as measured via automated counting of mKate2+ cells.

DETAILED DESCRIPTION

As used herein the term “EGFRvIII” or “vIII” refers to epidermal growth factor receptor variant III.

As used herein the term “EGFR” refers to human epidermal growth factor receptor. The term “wt EGFR”, “WT EGFR”, “EGFR WT” or “EGFR wt” are used interchangeably and refers to wild-type EGFR.

The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Unless specifically stated or obvious from context, as used herein the term “or” is understood to be inclusive and covers both “or” and “and”.

The term “and/or” where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other.

The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. The term “consisting of” is to be construed as close-ended. The term “consisting essentially of” when used in the context of CDR sequences means that the CDR sequence may be slightly (e.g., +/−1 or 2 aa) longer or shorter.

As used herein the term “native” with respect to a protein such as EGFRvIII or EGFR refers to the natural conformation of the protein and includes proteins that are properly folded and/or functional.

As used herein the term “denatured” with respect to a protein such EGFRvIII or EGFR refers to a protein that has lost its natural conformation and may entail for example, a loss in the tertiary and secondary structure.

As used herein, the term “antibody” encompasses monoclonal antibody, polyclonal antibody, humanized antibody, chimeric antibody, human antibody, domain antibody, multispecific antibody (e.g., bispecific antibodies such as for example a bi-specific T-cell engager) etc. The term “antibody” encompasses molecules that have a format similar to those occurring in nature (e.g., human IgGs, etc.).

As used herein the term “not able to significantly bind a peptide” means that the binding is between 0% and 15% of that observed for the non-mutated EGFRvIII peptide (SEQ ID NO:5).

As used herein the term “antigen-binding domain” refers to the domain of an antibody or of an antigen-binding fragment which allows specific binding to an antigen.

The antigen-binding domain of the present disclosure may comprise for example, at least one complementarity determining region of the antibody heavy chain. In accordance with the present disclosure, the antigen-binding domain may comprise at least two complementarity determining regions of the antibody heavy chain. Further in accordance with the present disclosure, the antigen-binding domain may comprise three complementarity determining regions of antibody heavy chain. The antigen-binding domain may comprise or also comprise at least one complementarity determining region of the antibody light chain. In accordance with the present disclosure, the antigen-binding domain may comprise at least two complementarity determining regions of the antibody light chain. Further in accordance with the present disclosure, the antigen-binding domain may comprise at least three complementarity determining regions of the antibody light chain. The antigen-binding domain may comprise three complementarity determining regions of the light chain and three complementarity determining regions of the heavy chain of the antibody.

An exemplary embodiment of a molecule comprising an antigen-binding domain is an antibody or an antigen-binding fragment thereof.

Another exemplary embodiment of a molecule comprising an antigen-binding domain is a chimeric antigen receptor.

Yet another exemplary embodiment of a molecule comprising an antigen-binding domain is a bi-specific T-cell engager.

Yet a further exemplary embodiment of a molecule comprising an antigen-binding domain is a bispecific killer cell engager.

Another exemplary embodiment of a molecule comprising an antigen-binding domain is a trispecific killer cell engager.

A further exemplary embodiment of a molecule comprising an antigen-binding domain is an antigen-binding fragment such as for example a single chain Fv. In accordance with the present disclosure, the single chain Fv comprises for example, the heavy chain variable region and the light chain variable region of an antibody that specifically binds to EGFRvIII. The heavy chain variable region and the light chain variable region of the antibody may be connected by a linker.

Linker sequences include for example, an amino acid sequence of at least 5 amino acids. Multimers of the pentapeptide G₄S (e.g., (Gly4Ser)_(n) where n is a positive integer of 1 or more) are often used in the engineering of scFvs. Those include for example, a 15-mer (G₄S)₃ found in some of the first scFv fragments (Huston et al., 1988), a 18-mer GGSSRSSSSGGGGSGGGG (Andris-Widhopf et al., 2011) and the 20-mer (G₄S)₄ (Schaefer et al., 2010). Many other sequences have been proposed, including sequences with added functionalities, e.g. an epitope tag or an encoding sequence containing a Cre-Lox recombination site (Sblattero & Bradbury, 2000) or sequences improving scFv properties, often in the context of particular antibody sequences. The linker of the present disclosure may have the sequence GGGSGGGGSGGGGS (SEQ ID NO:47).

Chimeric Antigen Receptors

The present disclosure relates to chimeric antigen receptors that specifically bind to EGFRvIII and nucleic acid encoding same. The chimeric antigen receptors of the present disclosure comprise an antigen-binding domain of an antibody that specifically binds to EGFRvIII.

The basic structure of chimeric antigen receptors has been described in the literature (e.g., Gacerez, A. T. et al., J Cell Physiol. 231(12):2590-2598 (2016), Sadelain, M. et al. Cancer Discovery, 3(4):388-98, (2013), Zhang, C. et al., Biomarker Research, 5:22 (2017)).

Chimeric antigen receptors have an extracellular region (or ectodomain) which comprises an antigen-binding domain and an intracellular region (or endodomain) which comprises the transmembrane domain and the intracytoplasmic domain which comprise intracellular signaling domains of immune response pathways or immune effector function (e.g., cytolytic activity, helper activity including secretion of cytokines).

The general structure of the chimeric antigen receptors of the present disclosure is composed of an antigen-binding domain, a transmembrane domain, an optional costimulatory domain and an intracellular signaling domain.

The antigen-binding domain is generally composed of an antibody's heavy chain variable region and light chain variable region connected via a linker and forming, for example, a single chain (e.g., scFv). The antibody used to generate the CAR construct is selected for its ability to specifically bind to epidermal growth factor receptor variant III (EGFRvIII) while not binding to wild-type EGFR.

The heavy chain variable region may be at the N-terminus of the polypeptide chain, followed by the linker and the light chain variable region. Alternatively, the light chain variable region may be at the N-terminus of the polypeptide chain, followed by the linker and the heavy chain variable region. In some instances, the single chain Fv may also comprises portions of the constant region.

Chimeric antigen receptors may also comprise a hinge region or spacer which connects the antigen-binding domain and the transmembrane domain. The spacer may allow a better presentation of the antigen-binding domain at the surface of the cell.

In accordance with the present disclosure, the spacer may be optional. Alternatively, the spacer may comprise for example, between 1 to 200 amino acid residues, typically between 10 to 100 amino acid residues and more typically between 25 to 50 amino acid residues. The spacer may originate from a human protein.

In accordance with the present disclosure, the spacer or hinge region may be, for example and without limitation a CD8 hinge (e.g., mouse, human CD8) or an IgG hinge (a human immunoglobulin hinge) or combination thereof.

An exemplary embodiment of a linker and hinge combination is provided in SEQ ID NO:81 where the hinge portion is from human CD8.

The transmembrane domain allows the extracellular region of the CAR to be anchored at the cell membrane. The transmembrane domain may be natural or synthetic and usually comprises hydrophobic amino acid residues. The transmembrane domain may be obtained from any naturally occurring protein having a transmembrane domain. The transmembrane domain is particularly selected for its ability to signal to the intracellular domain that the antigen-binding domain has bound to its target.

Exemplary embodiments of transmembrane domains include, for example and without limitation, the alpha, beta or CD3zeta chain of the T-cell receptor complex, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

In some embodiments, the transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB 1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMI, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, LyI08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C.

A particular embodiment of transmembrane domain is the transmembrane domain of CD28.

In accordance with the present disclosure, the chimeric antigen receptors may comprise one or more intracellular signaling domain derived from CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma RIIa, DAP10, or DAP12.

Chimeric antigen receptors may also optionally comprise at least one costimulatory domain.

In accordance with the present disclosure, the costimulatory domain may be, for example, from CD28, CD27, 4-1BB, OX40, CD7, B7-1 (CD80), B7-2 (CD86), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LyI08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D or a combination thereof.

In order to be targeted to the secretory pathway, the chimeric antigen receptor may also comprise a signal peptide such as, for example, a signal peptide of CD28 or any other signal peptide suitable for immune cells. The signal peptide is cleaved (cleavable).

The antigen-binding fragments of the present disclosure may comprise human or humanized framework amino acid sequences.

Antibodies or Antigen-Binding Fragments

Typically, an antibody is constituted from the pairing of two light chains and two heavy chains. Different antibody isotypes exist, including IgA, IgD, IgE, IgG and IgM. Human IgGs are further divided into four distinct sub-groups namely; IgG1, IgG2, IgG3 and IgG4. Therapeutic antibodies are generally developed as IgG1 or IgG2.

In an exemplary embodiment, the antibody or antigen-binding fragment of the present disclosure may comprise, for example, a human IgG1 constant region. In another exemplary embodiment, the antibody or antigen-binding fragment of the present disclosure may comprise, for example, a human IgG2 constant region.

The light chain and heavy chain of human antibody IgG isotypes each comprise a variable region having 3 hypervariable regions named complementarity determining regions (CDRs). The light chain CDRs are identified herein as CDRL1 or L1, CDRL2 or L2 and CDRL3 or L3. The heavy chain CDRs are identified herein as CDRH1 or H1, CDRH2 or H2 and CDRH3 or H3. Complementarity determining regions are flanked by framework regions (FR) in the order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. CDRs may be identified using for example, the Kabat and Chotia definitions (Kabat, J. Immunol. 1991, Chotia and Lesk 1987). However, others (Abhinandan and Martin, 2008) have used modified approaches based loosely on Kabat and Chotia resulting in the delineation of shorter CDRs. Lefranc also discloses the IMGT numbering scheme for CDRs (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999)).

The overall binding affinity of the antibody or antigen-binding fragment thereof is often dictated by the sequence of the CDRs. The framework regions may also play a role in the proper positioning and alignment in three dimensions of the CDRs for optimal antigen-binding.

As used herein an “antigen-binding fragment” refers to a fragment of an antibody that may be obtained by enzymatic digestion of an antibody, by recombinant DNA technology and the like. Antigen-binding fragments thereof of the present disclosure encompass molecules having an antigen-binding domain comprising amino acid residues that confer specific binding to an antigen (e.g., one or more CDRs).

Examples of antigen-binding fragments encompassed by the present disclosure include (i) a Fab fragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1) domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_(H) and C_(H1), domains; (iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a single arm of an antibody (e.g. scFv), (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a V_(H) domain; and (vi) an isolated complementarity determining region (CDR), e.g., V_(H) CDR3.

Although the two domains of the Fv fragment, V_(L) and V_(H), are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single polypeptide chain in which the V_(L) and V_(H) regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

Single chain antibodies (e.g., single domain), diabody, minibody, nanobody and the like are encompassed within the term “antigen-binding fragment”. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for activity in the same manner as for intact antibodies.

Particular embodiments of antigen-binding fragments may include for example, a scFv, a Fab, a Fab′ or a (Fab′)₂.

The term “humanized antibody” encompasses fully humanized (i.e., frameworks are 100% humanized) and partially humanized sequences (e.g., at least one variable region contains one or more amino acids from a human antibody, while other amino acids are amino acids of a non-human parent antibody). Typically, a “humanized antibody or antigen-binding fragment” contains CDRs of a non-human parent antibody (e.g., mouse, rat, rabbit, non-human primate, etc.) and frameworks that are identical to those of a natural human antibody or of a human antibody consensus. In such instance, those “humanized antibodies or antigen-binding fragments” are characterized as fully humanized. A “humanized antibody or antigen-binding fragment” may also contain one or more amino acid substitutions that have no correspondence to those of the human antibody or human antibody consensus. Such substitutions include, for example, back-mutations (e.g., re-introduction of non-human amino acids) that may preserve the antibody characteristics (e.g., affinity, specificity etc.). Such substitutions are usually in the framework region. A “humanized antibody or antigen-binding fragment” usually also comprise a constant region (Fc) or a portion thereof which is typically that of a human antibody. Typically, the constant region of a “humanized antibody or antigen-binding fragment” is identical to that of a human antibody. A humanized antibody may be obtained by CDR grafting (Tsurushita et al, 2005; Jones et al, 1986; Tempest et al, 1991; Riechmann et al, 1988; Queen et al, 1989). Such antibody is considered as fully humanized.

The term “chimeric antibody” refers to an antibody having a constant region from an origin distinct from that of the parent antibody. The term “chimeric antibody” encompasses antibodies having a human constant region. Typically, a “chimeric antibody” is composed of variable regions originating from a mouse antibody and of human constant regions.

The term “hybrid antibody” refers to an antibody comprising one of its heavy or light chain variable region (its heavy or light chain) from a certain type of antibody (e.g., humanized) while the other of the heavy or light chain variable region (the heavy or light chain) is from another type (e.g., murine, chimeric).

Antibodies and/or antigen-binding fragments of the present disclosure may originate, for example, from a mouse, a rat or any other mammal or from other sources such as through recombinant DNA technologies. Antibodies or antigen-binding fragment of the present disclosure may include for example, a synthetic antibody, a non-naturally occurring antibody, an antibody obtained following immunization of a non-human mammal etc.

Antibodies or antigen-binding fragments thereof of the present disclosure may be isolated and/or substantially purified.

Variants

The present disclosure also encompasses variants of the antigen-binding agents described herein. Variant of the present disclosure include those having a variation in their amino acid sequence, e.g., in one or more CDRs, in one or more framework regions and/or in the constant region. Variant included in the present disclosure are those having, for example, similar or improved binding affinity in comparison with an original antigen-binding agent.

Exemplary variants encompassed by the present disclosure are those which may comprise an insertion, a deletion or an amino acid substitution (conservative or non-conservative). These variants may have at least one amino acid residue in its amino acid sequence removed and a different residue inserted in its place.

More particularly, variants encompassed by the present disclosure include those having a light chain variable region and/or a heavy chain variable region having at least 80% sequence identity with the light chain variable region and/or a heavy chain variable region of the antibodies or antigen-binding fragments, CARs, BiTEs, BiKEs or TriKEs disclosed herein. The CDRs of the variants of the present disclosure may be identical to those of the antibodies or antigen-binding fragments, CARs, BiTEs, BiKEs or TriKEs disclosed herein.

Also encompassed by the present disclosure are variants having CDRs amino acid residues that are identical and framework regions that are at least 80% sequence identical to those of the antigen-binding domain, antibody or antigen-binding fragment disclosed herein.

Conservative substitutions may be made by exchanging an amino acid residue (of a CDR, variable chain, framework region or constant region, etc.) from one of the groups listed below (group 1 to 6) for another amino acid of the same group.

Other exemplary embodiments of conservative substitutions are shown in Table A.

(group 1) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile)

(group 2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr)

(group 3) acidic: Aspartic acid (Asp), Glutamic acid (Glu)

(group 4) basic: Asparagine (Asn), Glutamine (Gin), Histidine (His), Lysine (Lys), Arginine (Arg)

(group 5) residues that influence chain orientation: Glycine (Gly), Proline (Pro); and

(group 6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe)

Non-conservative substitutions will entail exchanging a member of one of these groups for another.

TABLE A Amino acid substitution Original Exemplary Conservative residue substitution substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg, Asp Gln Asp (D) Glu, Asn Glu Cys (C) Ser, Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp, Gln Asp Gly (G) Ala Ala His (H) Asn, Gln, Lys, Arg, Arg Ile (I) Leu, Val, Met, Ala, Leu Phe, norleucine Leu (L) Norleucine, Ile, Val, Ile Met, Ala, Phe Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Leu norleucine

Percent identity is indicative of amino acids which are identical in comparison with the original peptide and which may occupy the same or similar position. Percent similarity will be indicative of amino acids which are identical and those which are replaced with conservative amino acid substitution in comparison with the original peptide at the same or similar position.

Generally, the degree of similarity and identity between variable chains has been determined herein using the Blast2 sequence program (Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250) using default settings, i.e., blastp program, BLOSUM62 matrix (open gap 11 and extension gap penalty 1; gapx dropoff 50, expect 10.0, word size 3) and activated filters.

Variants of the present disclosure therefore comprise those which may have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an original sequence or a portion of an original sequence.

Nucleic Acids, Vectors and Cells

As used herein, the term “nucleic acid” refers to RNA, DNA, cDNA and the like.

The present disclosure encompasses nucleic acids capable of encoding any of the CDRs, light chain variable regions, heavy chain variable regions, light chains, heavy chains, scFvs antibodies or antigen-binding fragments, CARs, BiTEs, BiKEs or TriKEs or variants described herein.

Nucleic acid molecules encoding CARs, BiTEs, BiKEs or TriKEs are introduced into immune cells where they are expressed. Nucleic acids encoding CARs, BiTEs, BiKEs or TriKEs may be delivered by transduction systems such as for example, using viral systems (from lentiviruses, adenoviruses, adeno-associated viruses, etc.) or non-viral systems (e.g., transposon). Exemplary system involving transposons includes the Sleeping Beauty transposon system and the piggyBac transposon system. Gene editing system may also be used including, without limitation, the CRISPR/cas system, the Zinc finger nuclease system, the Transcription Activator-Like Effector Nucleases (TALENs) system. Nucleic acids (e.g., RNA or DNA) may be delivered by other means such as by liposomes, naked DNA, polymers, electroporation etc.

Due to the inherent degeneracy of the genetic code, other nucleic acid sequences that encode the same amino acid sequence may be produced and used to express the antibody or antigen-binding fragments thereof of the present disclosure. The nucleotide sequences may be engineered using methods generally known in the art in order to alter the nucleotide sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.

In yet another aspect, the present disclosure relates to a vector or vectors comprising the nucleic acids described herein.

In accordance with the present disclosure, the vector or vectors may be an expression vector(s).

Further in accordance with the present disclosure, the vector may be a viral vector. Exemplary embodiments of viral vectors include lentiviral vectors, adenoviral vectors or adeno-associated viral vectors.

The expression vector usually contains the elements for transcriptional and translational control of the inserted coding sequence in a particular host. These elements may include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5′ and 3′ un-translated regions. Exemplary embodiments of promoter used to drive CAR construct expression in T cells includes the EF1 a promoter. Other exemplary embodiments of promoters include constitutively active viral promoters such as for example, the immediate early cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR), Rous sarcoma virus (RSV) promoter.

Methods that are well known to those skilled in the art may be used to construct such expression vectors. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

In order to make the antibodies or antigen-binding fragments of the present disclosure, a vector or a set of vectors expressing the light chain and heavy chain are introduced into a cell.

The present disclosure encompasses vectors or a set of vectors where the light chain variable region and the heavy chain variable region of the antibody or antigen-binding fragment thereof are encoded by the same nucleic acid molecule (e.g., same vector) or by separate nucleic acid molecules (e.g., separate vectors).

In another aspect the present disclosure relates to an isolated cell which may comprise the nucleic acids, vectors, antibodies or antigen-binding fragment, CARs, BiTEs, BiKEs or TriKEs described herein.

Yet another aspect of the present disclosure relates to an isolated host cell which expresses the antibody or antigen-binding fragment, CAR, BiTE, BiKE or TriKE of the present disclosure. The isolated host cell for CAR, BiTE, BiKEs or TriKE expression may be an immune cell such as for example and without limitation, T cells, Natural Killer (NK) cells, cytotoxic T cells, or regulatory T cells.

The present disclosure also relates to a cell population engineered to express the CAR, BiTE, BiKE or TriKE of the present disclosure. The cell population may be homogenous or heterogenous. In accordance with the present disclosure, the cell population may comprise T cells (CD4+ T-cells, CD8+ T-cells or a combination thereof), Natural Killer (NK) cells, cytotoxic T cells, regulatory T cells, and combinations thereof. In accordance with the present disclosure the cell population may be autologous. In accordance with the present disclosure the cell population may be allogenic.

As used herein the term “autologous” refers to material derived from the same individual.

The term “allogeneic” refers to material derived from a different subject of the same species but that is genetically distinct.

As used herein, the term “heterogenous” with reference to a cell population means that the cell population either express different chimeric antigen receptors or that the cell population comprises different types of cells.

As used herein, the term “homogenous” with reference to a cell population means that the cell population either express the same chimeric antigen receptors or that the cell population comprises the same type of cells.

The present disclosure also encompasses complement of the nucleic acids disclosed herein. It is to be understood herein that nucleic acid molecules comprising at least a portion complementary to the nucleic acid sequence disclosed herein are also encompassed by the present disclosure. Such complementary nucleic acid molecules may be used, for example, for gene amplification or detection of the nucleic acid molecule of the present disclosure and include probes or primers.

Production of Cells Expressing Chimeric Antigen Receptors

Methods of manufacturing or of producing immune cells expressing chimeric antigen receptors, bi-specific T-cell engagers, bispecific killer cell engagers or trispecific killer cell engagers.

Immune cells of human origin may particularly be used to generate the CAR-expressing cell population of the present disclosure. The immune cells may comprise T-cells (e.g., CD4⁺ or CD8⁺), Natural Killer cells, cytotoxic T-cells, regulatory T-cells, and combinations thereof. Particularly contemplated are T-cells or NK cells.

Immune cells are first isolated from a subject by various methods known to a person skilled in the art. For example, peripheral blood mononuclear cells (PBMCs) may be isolated from a subject by leukaphoresis. T-cells may be enriched and washed to separate them from the leukocytes. The different T cell subsets may be separated using beads conjugated with specific antibody or markers. CAR-T cells are often generated from the CD3⁺ population. T-cells are activated by various methods including for example, by antigen-presenting cells, with specific antibodies and the like (methods reviewed in Wang, X et al., Molecular Therapy—Oncolytics, 3:16015, 2016).

The method may comprise introducing the isolated nucleic acid molecule or the vector disclosed herein into the immune cells such that the nucleic acid integrates into the genome of the cell. The cells may be transduced using viral vectors or by other means and expanded. For example, lentiviral vectors are used to transduce immune cells with CAR-expression by exposing cells directly to viral vector containing supernatant media overnight. Cells are then assessed for transduction using a fluorescent marker included in the CAR-expressing lentiviral backbone.

Following purification and quality control steps, CAR-expressing immune cells are provided to a subject in need. The subject in need may be, for example, the initial donor of the immune cells.

Production of the Antibodies or Antigen-Binding Fragments in Cells

The antibodies that are disclosed herein can be made by a variety of methods familiar to those skilled in the art including hybridoma methodology or recombinant DNA methods.

Conventional hybridoma technology entails immunizing a rodent with an antigen, isolating and fusing spleen cells with myeloma cells lacking HGPRT expression and selecting hybrid cells by hypoxanthine, aminopterin and thymine (HAT) containing media. Hybridoma are screened to identify those producing antibodies that are specific for a given antigen. The hybridoma is expanded and cloned. The nucleic acid sequence of the light chain and heavy chain variable regions is obtained by standard sequencing methodology and expression vectors comprising the light chain and heavy chain nucleic acid sequence of an antibody are generated.

For recombinant expression of antibodies, host cells are transformed with a vector or a set of vectors comprising the nucleic acid sequence of the light chain and heavy chain of the antibody or antigen-binding fragment thereof (on the same vector or separate vectors).

For long-term production of recombinant proteins in mammalian systems, stable expression in cell lines may be effected. For example, nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein may be transformed into cell lines using expression vectors that may contain viral origins of replication and/or endogenous expression elements and a selectable or visible marker gene on the same or on a separate vector. The disclosure is not to be limited by the vector or host cell employed. In certain embodiments of the present disclosure, the nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein may each be ligated into a separate expression vector and each chain expressed separately. In another embodiment, both the light and heavy chains able to encode any one of a light and heavy immunoglobulin chains described herein may be ligated into a single expression vector and expressed simultaneously.

Immunological methods for detecting and measuring the expression of polypeptides are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), fluorescence activated cell sorting (FACS) or flow cytometry. Those of skill in the art may readily adapt these methodologies to the present disclosure.

Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., Chinese Hamster Ovary (CHO), HeLa, MDCK, HEK293, and W138) are available commercially and from the American Type Culture Collection (ATCC) and may be chosen to ensure the correct modification and processing of the expressed polypeptide.

Typically, antibody or antigen-binding fragments thereof are produced in CHO cells, NSO murine myeloma cells, PER.C6® human cells.

The present disclosure relates to a method of making an antibody or an antigen-binding fragment thereof comprising expressing the light chain and heavy chain of the antibody or antigen-binding fragment of the present disclosure in cultured cells.

The method may further comprise purifying or isolating the antibody or antigen-binding fragment of the present disclosure. The method may also further comprise conjugating the antibody or antigen-binding fragment of the present disclosure to a cargo molecule such as a therapeutic or detectable moiety.

Antibody Conjugates

The antibody or antigen-binding fragment thereof of the present disclosure may be linked to a cargo molecule. Exemplary embodiments of cargo molecules include without limitation a therapeutic moiety a detectable moiety, a polypeptide (e.g., peptide, enzyme, growth factor), a polynucleotide, liposome, nanoparticle, nanowire, nanotube, quantum dot, etc.

More particularly, the antibody or antigen-binding fragment thereof of the present disclosure may be conjugated with a therapeutic moiety. The therapeutic moiety is usually attached to the antibody via a linker which may be cleavable or non-cleavable.

Included amongst the list of therapeutic moiety are cytotoxic agents, cytostatic agents, anti-cancer agents (chemotherapeutics) and radiotherapeutics (e.g. radioisotopes).

Exemplary embodiments of cytotoxic agents include, without limitation, alpha-amanitine, cryptophycin, duocarmazine, duocarmycin, chalicheamicin, deruxtecan, pyrrolobenzodiazepine (PBD), dolastatins, pseudomonas endotoxin, ricin, auristatins (e.g., monomethyl auristatin E, monomethyl auristatin F), maytansinoids (e.g., mertansine) and analogues.

Exemplary embodiments of radiotherapeutics include without limitation, Yttrium-90, Scandium-47, Rhenium-186, Iodine-131, Iodine-125, and many others recognized by those skilled in the art (e.g., lutetium (e.g., Lu¹⁷⁷), bismuth (e.g., Bi²¹³), copper (e.g., Cu⁶⁷)), astatine-211 (211At) actinium 225 (Ac²²⁵).

Exemplary embodiments of chemotherapeutics include, without limitation, 5-fluorouracil, adriamycin, irinotecan, taxanes, carboplatin, cisplatin, etc.

The antibody or antigen-binding fragment of the present disclosure may also be conjugated with a detectable moiety (i.e., for detection or diagnostic purposes).

A “detectable moiety” comprises agents detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical and/or other physical means. A detectable moiety may be coupled either directly and/or indirectly (for example via a linkage, such as, without limitation, a DOTA or NHS linkage) to antibodies and antigen-binding fragments thereof of the present disclosure using methods well known in the art. A wide variety of detectable moieties may be used, with the choice depending on the sensitivity required, ease of conjugation, stability requirements and available instrumentation. A suitable detectable moiety include, but is not limited to, a fluorescent label, a radioactive label (for example, without limitation, ¹²⁵I, In¹¹¹, Tc⁹⁹, I¹³¹ and including positron emitting isotopes for PET scanner etc), a nuclear magnetic resonance active label, a luminescent label, a chemiluminescent label, a chromophore label, an enzyme label (for example and without limitation horseradish peroxidase, alkaline phosphatase, etc.), quantum dots and/or a nanoparticle. Detectable moiety may cause and/or produce a detectable signal thereby allowing for a signal from the detectable moiety to be detected.

Pharmaceutical Compositions

The present disclosure relates to a pharmaceutical composition which may comprise the antibodies or antigen binding fragments thereof, BiTEs, BiKEs or TriKEs of the present disclosure. The pharmaceutical composition may comprise, for example, diluents and/or other components such as immunomodulatory antibodies including but not limited to immune checkpoint blocking antibodies, cytokines or chemokines.

The present disclosure relates to a pharmaceutical composition which may comprise a cell population engineered to express CARs, BiTEs, BiKEs or TriKEs of the present disclosure. The pharmaceutical composition may comprise, for example, diluents and/or other components such as immunomodulatory antibodies including but not limited to immune checkpoint blocking antibodies, cytokines or chemokines.

The present disclosure also relates to pharmaceutical compositions comprising the antibodies or antigen-binding fragments (conjugated or not) disclosed herein.

In addition to the active ingredients, a pharmaceutical composition may contain pharmaceutically acceptable carriers comprising without limitation, water, PBS, salt solutions, gelatins, oils, alcohols, and other excipients and auxiliaries that facilitate processing of the active compounds into preparations that may be used pharmaceutically. In instances where the pharmaceutical composition comprises live cells (e.g., human NK cell lines) the preparation may be irradiated. Pharmaceutical compositions may also contain, without limitation; dextran, dextrose, dimethylsulfoxide (DMSO), human serum albumin, PlasmaLyte A™, sodium chloride

As used herein, “pharmaceutical composition” means therapeutically effective amounts of the agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers. A “therapeutically effective amount” as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts). Solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens, dimethylsulfoxide), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the disclosure are particulate compositions coated with polymers (e.g., poloxamers or poloxamines).

Other embodiments of the compositions of the disclosure incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, oral, vaginal, rectal routes. In one embodiment the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitonealy, intraventricularly, intracranially and intratumorally.

Further, as used herein “pharmaceutically acceptable carrier” or “pharmaceutical carrier” are known in the art and include, but are not limited to, 0.01-0.1 M or 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.

For any compound, the therapeutically effective dose may be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans. These techniques are well known to one skilled in the art and a therapeutically effective dose refers to that amount of active ingredient that ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating and contrasting the ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population) statistics. Any of the therapeutic compositions described above may be applied to any subject in need of such therapy, including, but not limited to, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and humans.

Pharmaceutical compositions comprising cells may be administered by infusion (e.g., by intravenous route, intracerebral injection or other routes). Pharmaceutical compositions comprising antibodies of the present disclosure may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

In certain instances, the CAR cell population may be administered concurrently in combination with other treatments given for the same condition including for example anti-cancer agents. Exemplary embodiments of anti-cancer agents include for example and without limitation, therapeutic antibodies, immunomodulators (immune checkpoint blocking antibodies), anti-mitotics (e.g., taxanes), platinum-based agents (e.g., cisplatin), DNA damaging agents (eg. Doxorubicin) and other anti-cancer therapies that are known to those skilled in the art.

Additional aspects of the disclosure relate to kits which may include vial(s) containing one or more nucleic acid encoding the CARs, BiTEs, BiKEs, TriKEs or antibodies or antigen-binding fragments described herein.

Methods of Use

Aspects of the disclosure comprise administering antibodies or antigen binding fragments thereof, CAR, BiTE, BiKE or TriKE molecules to a subject in need.

Other aspects of the disclosure comprise administering immune cells engineered to express the CAR, BiTE, BiKE or TriKE molecules to a subject in need.

The CAR, BiTE, BiKE or TriKE constructs of the present disclosure may be used to re-target engineered immune cells towards EGFRvIII-positive tumors.

The engineered immune cells may be administered to a subject in need.

In accordance with an aspect of the present disclosure, immune cells are isolated from the subject, engineered to express the CAR, BiTE, BiKE or TriKE construct and re-administered to the same subject.

As used herein the term “subject” encompasses humans and animals such as non-human primates, cattle, rabbits, mice, rats, sheep, goats, horses, birds, etc. The term “subject” particularly encompasses humans.

Subjects in need which would benefit from treatment include humans having tumor cells expressing EGFRvIII. More particularly, the immune cells engineered to express the CAR, BiTE, BiKE or TriKE construct disclosed herein may be administered to a subject suspected of having glioblastoma multiforme (GBM). Subjects in need also encompass those having or suspected of having carcinomas, such as breast carcinoma, ovarian carcinoma, prostate carcinoma, or non-small cell lung carcinomas.

The term “treatment” for purposes of this disclosure refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. Particularly, subjects in need include subjects with an elevated level of one or more cancer markers.

The present disclosure more particularly relates to a method of treating a subject having or suspected of having cancer by administering a cell population expressing the chimeric antigen receptor or the antibody or antigen-binding fragment thereof disclosed herein.

The cell population expressing the chimeric antigen receptor, or the antibody or antigen-binding fragment thereof may be administered as a pharmaceutical composition either alone or in combination with other anti-cancer drugs.

Other aspects of the disclosure relate to a method for detecting EGFRvIII, the method may comprise contacting a cell expressing EGFRvIII, or a sample (biopsy, a body fluid such as serum, plasma, urine etc.) comprising or suspected of comprising EGFRvIII with the antibody or antigen-binding fragments described herein and measuring binding. The sample may originate from a mammal (e.g., a human) which may have cancer (e.g., glioblastoma multiforme or carcinoma) or may be suspected of having such cancer. The sample may be a tissue sample obtained from the mammal or a cell culture supernatant.

In accordance with the disclosure the sample may be a serum sample, a plasma sample, a blood sample or ascitic fluid obtained from the mammal.

Further scope, applicability and advantages of the present disclosure will become apparent from the non-restrictive detailed description given hereinafter. It should be understood, however, that this detailed description, while indicating exemplary embodiments of the disclosure, is given by way of example only, with reference to the accompanying drawings.

EXAMPLES Example 1: Generation of EGFRvIII Specific Monoclonal Antibodies

Monoclonal antibodies (mAb) against EGFRvIII were generated by immunizing mice with the extracellular domain of recombinant proteins.

Immunizations

Mice were bled (pre-immune serum) and injected intraperitoneally and subcutaneously with 100 μg of recombinant EGFRvIII protein emulsified in Titermax adjuvant (Cedarlane Labs, Burlington, ON) at day 0 and in PBS without adjuvant at day 22. Blood was collected in microvette CB 300Z (Sarstedt, Montreal, QC) at day 29, and serum was stored at −20° C. until further use.

ELISA (Serum Titer Determination)

Pre- and post-immune sera titers of animals were assessed by ELISA on recombinant EGFRvIII protein. Unless otherwise stated, all incubations were performed at room temperature. Briefly, half-area 96-well plates (Costar #3690) were coated with 25 μl per well of immunogen at 5 μg/ml in PBS and incubated overnight at 4° C. Microplates were washed three times in PBS and blocked for 30 min with PBS containing 1% bovine serum albumin (BSA, Sigma Cat#A7030). Blocking buffer was removed and 25 μl of serial dilutions of sera samples were added. After a 2-h incubation, microplates were washed 4 times with PBS-TWEEN™ 20 0.05% and 25 μl of a 1/5,000 dilution of alkaline phosphatase conjugated F(ab′)′₂ goat anti-mouse IgG (H+L, #115-056-062, Jackson Immunoresearch, Cedarlane, Burlington, ON) in blocking buffer was added. After a 1-h incubation, microplates were washed 4 times and 25 μl of p-nitrophenyl phosphate (pNPP) substrate (Sigma-Aldrich Canada Co., Oakville, ON) at 1 mg/ml in carbonate buffer at pH 9.6 was added and further incubated for 30 min. Absorbance was read at 405 nm using a SpectraMax 340 PC plate reader (Molecular Devices, Sunnyvale, Calif.). All pre-immune bleeds were negative and all post-immune bleeds were very strong (titer above 1/51200) on recombinant protein.

Generation of Hybridomas

Mice received a final boost of 100 ug of recombinant EGFRvIII protein and their spleen was harvested 3 to 4 days later. All manipulations were done under sterile conditions. Spleen cells were harvested in Iscove's Modified Dulbecco's medium (IMDM, Gibco Cat. #31980-030) and fused to NSO myeloma cell line using electrofusion protocol.

Electrofusion Protocol

Spleen cells and myeloma cells were washed separately in IMDM. Cells were washed in Isoosmolar buffer (Eppendorf cat#4308070536), then in Cytofusion Medium C (BTX cat#47-0001). Myeloma and lymphocytes were mixed together at a 1:1 ratio and fused using an ECM 2001 Cell Fusion System (BTX, Harvard Bioscience Inc.) following manufacturer's instructions.

Following fusion, cells were suspended at a concentration of 2-4×10⁵ input myeloma cells per ml in HAT selection medium (IMDM containing 20% heat inactivated FBS, penicillin-streptomycin (Sigma Cat#P7539), 1 ng/ml mouse IL-6 (Biolegend Cat#575706), HAT media supplement (Sigma Cat#H0262) and L-glutamine (Hy-Clone Cat#SH30034.01) and incubated at 37° C., 5% CO₂. The next day, hybridoma cells were washed and suspended at a concentration of 2-5'10⁵ input myeloma cells per ml in semi-solid medium D (StemCell Technologies Cat.#03804) supplemented with 5% heat inactivated FBS, 1 ng/ml mouse IL-6 and 10 μg/ml FITC-F(ab′)₂ Goat anti-mouse IgG Fc gamma specific (Jackson # 115-096-071). The cell mixture was plated in Omnitray dish (Nunc cat#242811) and further incubated for 6-7 days at 37° C., 5% CO₂. Fluorescent secretor clones were then transferred using a mammalian cell clone picker (ClonepixFL™, Molecular Devices) into sterile 96-w plates (Costar #3595) containing 200 μl of IMDM supplemented with 20% heat inactivated FBS, penicillin-streptomycin, 1 ng/ml mouse IL-6, HT media supplement (Sigma Cat# H0137) and L-glutamine and incubated for 2-3 days at 37° C., 5% CO₂.

Five thousand (5000) hybridoma supernatants from seven (7) fusion experiments were screened by ELISA using recombinant EGFRvIII or EGFR wild-type proteins to detect specific binders. To this end, half-area 96-well plates (Costar #3690) were coated with 25 μl per well of immunogen at 5 μg/ml in PBS and incubated overnight at 4° C. Microplates were washed three times in PBS and blocked for 30 min with PBS containing 1% bovine serum albumin (BSA, Sigma Cat#A7030). Blocking buffer was removed and 25 μl of hybridoma supernatant were added. After a 2-h incubation, microplates were washed 4 times with PBS-TWEEN™ 20 0.05% and 25 μl of a 1/5,000 dilution of alkaline phosphatase conjugated F(ab′)′₂ goat anti-mouse IgG (Fc specific, #115-056-071, Jackson Immunoresearch, Cedarlane, Burlington, ON) in blocking buffer was added. After a 1-h incubation, microplates were washed 4 times and 25 μl of p-nitrophenyl phosphate (pNPP) substrate (Sigma-Aldrich Canada Co., Oakville, ON) at 1 mg/ml in carbonate buffer at pH 9.6 was added and further incubated for one hour at 37° C. Absorbance was read at 405 nm using a SpectraMax 340 PC plate reader (Molecular Devices, Sunnyvale, Calif.).

ELISA positive antibodies were selected and further characterized by flow cytometry on U87MG cells overexpressing wild-type human EGFR (U87MG-EGFR WT) or human EGFRvIII (U87MG-EGFRvIII) to confirm their specificity. To this end, 15-ml supernatant from each positive clone was produced.

In order to compare our results with previous studies, additional monoclonal antibodies were used including the 13.1.2 antibody which is specific to EGFRvIII mutation (Hamblett K. J, et al., 2015; U.S. Pat. No. 7,736,644) and the 225 monoclonal antibody which is a murine antibody recognizing both wild-type human EGFR and human EGFRvIII proteins (Mendelson et al.; 2015).

Example 2: Cell Surface Binding by Flow Cytometry

For specificity analysis, several cell lines were used including human glioblastoma cell lines U87MG overexpressing wild-type human EGFR (a.k.a., U87MG-EGFR WT or U87 WT) and U87MG overexpressing human EGFRvIII (Δ2-7 deletion mutation of EGFR a.k.a. U87MG-EGFRvIII or U87vIII).

The binding properties of the anti-EGFRvIII monoclonal antibodies selected in Example 1 were assessed by flow cytometry on human glioblastoma cell lines U87MG overexpressing wild-type EGFR and U87MG overexpressing EGFRvIII mutation.

Briefly, cells overexpressing full length wt EGFR or EGFRvIII were obtained from the laboratory of W. Cavanee (Ludwig Institute for Cancer Research, University of California at San Diego). Cells were grown in DMEM high glucose medium containing 10% FBS and 400 μg/m1 G418. Prior to analysis, cells were plated such that they were not more than 80% confluent on the day of analysis. Unless otherwise stated, all media are kept are 4° C. and all incubations are performed on wet ice. Cells were washed in PBS and harvested by the addition of cell dissociation buffer (Sigma), centrifuged and resuspended in complete medium at a cell density of 2×10⁶ cells/mL. Fifty μL/well of cells are distributed in a polypropylene v-bottom 96 well plate and equal volume of hybridoma supernatant were added and incubated for 2 hours. Cells were washed twice by centrifugation and further incubated with a FITC labeled F(ab′)2 goat anti-mouse antibody (Fc specific, #115-096-071, Jackson Immunoresearch, Cedarlane, Burlington, ON) for an hour. Cells were washed and resuspended in medium containing Propidium iodide to exclude dead cells from analysis. Samples were filtered through a 60 μm nylon mesh filter plate (Millipore, Ireland) to remove cell aggregates. Flow cytometry analyses were performed on 2,000 viable single-cells events gated on forward scattering, side scattering parameters and propidium iodide dye exclusion using a BD-LSR Fortessa flow cytometer (Becton-Dickinson Biosciences, CA, USA) and a standard filter set using BD FACSDiva™ acquisition software, according to manufacturer's instructions.

Cells were stained with either negative control anti-GFP 3E6 monoclonal antibody supernatant (open histograms) or tested hybridoma supernatant (grey histograms). Specific binding was reflected by the increase in the mean fluorescent intensity of antibody binding to U87cells expressing EGFRvIII but not wild-type EGFR.

Out of the 36 positive cell based binding antibodies derived from 7 independent fusion experiments, we chose to further study three hybridoma supernatants, whose binding was found to be specific for EGFRvIII overexpressing U87MG cells, including F265-5B7 (FIG. 1A) referred to herein also as 5B7, F269-3D12 (FIG. 16) referred to herein also as 3D12 and F271-1D2 (FIG. 1C) referred to herein also as 1 D2. Data obtained for the 225 antibody (ATCC HB-8508) and the 13.1.2 antibody are shown in FIG. 1D and 1E respectively.

Example 3: Evaluation of Binding on Purified Denatured Antigen

To evaluate if monoclonal antibodies bind to a conformational epitope, an ELISA analysis on native and denatured recombinant human wild-type EGFR and EGFRvIII proteins were performed. The 13.1.2 antibody) and the 225 antibody were used as controls. Antigens at 1-2 mg/ml were denatured by incubation at 95° C. for 5 min in PBS containing DTT at 40 mM final concentration. They were then incubated on ice for 5 min and diluted at their final coating concentration for ELISA purpose.

mAbs were purified using HiTrap ProteinG HP 1 mL columns GE Healthcare cat no. 17-0404-01 and desalted using Zeba-spin desalting columns 5 mL (Pierce) pre-equilibrated in PBS and filter sterilized through 0.22 μM membrane (Millipore). The final concentration of the antibody solutions was determined using a Nano-drop 2000 (ThermoScientific), using IgG as sample type. ELISA was performed as described above (serum titer determination) using 25 μl of mAb supernatant (Exp 1) or purified mAb at 1 μg/ml (Exp 2).

Table 1 shows ELISA results (n=2) of different mAb clones assessed on recombinant human EGFRvIII or wild-type EGFR, in native or denatured conditions. As expected, the 225 antibody binds to both wild-type EGFR and EGFRvIII under native conditions only. The 13.1.2 antibody binds to EGFRvIII in native and denatured conformations, but not to EGFR wild-type native or denatured. All other mAbs binds to EGFRvIII in native and denatured conformations.

TABLE 1 Binding on native or denatured recombinant proteins by ELISA EGFRvIII EGFRwt Native Denatured Native Denatured Clone Exp 1 Exp 2 Exp 1 Exp 2 Exp 1 Exp 2 Exp 1 Exp 2 5B7 1.144 0.874 1.674 0.623 0.008 0.002 0.046 0.000 3D12 1.173 1.125 1.859 0.517 0.095 0.011 0.045 0.001 1D2 1.491 0.935 2.553 1.054 0.002 0.004 ND 0.000 225 1.081 1.496 0.001 0.004 1.204 1.320 −0.010 0.002 13.1.2 1.097 1.671 1.914 1.705 0.011 0.003 0.003 0.005 mIgG1 13.1.2 1.291 1.487 2.094 1.495 0.009 0.003 0.007 0.002 mIgG2a Neg ctrl ND −0.001 ND 0.002 ND 0.002 ND 0.000 mouse mAb ND: not determined

Selected hybridoma were recloned by limiting dilution to ensure their monoclonality.

Example 4. Epitope Mapping by Yeast Surface Display

The yeast surface display method (Feldhaus M J et al., 2003 Nat Biotechnol. 2003 February;21(2):163-70) was used to map the epitopes of our collection of monoclonal antibodies against EGFRvIII. This technique allows cloned protein or peptide of choice to be expressed and displayed at cell surface through covalent linkage to cell wall. The displayed protein/peptide can be interrogated for antibody binding.

A total of 36 different human EGFRvIII fragments of variable size from 10 to 414 as indicated in FIG. 3 were cloned into the pPNL6 vector (obtained from The Pacific Northwest National Laboratory, USA) as fusion proteins to be expressed and displayed as Aga2-HA-hEGFRvIII-MYC. The displayed human EGFRvIII fragments were used to identify the smallest fragment required for the binding of each anti-EGFRvIII monoclonal antibodies.

Assessment of the binding of anti-EGFRvIII monoclonal antibodies to the fusion proteins expressed on yeast cell surface was done by flow cytometry analysis. Yeast cells were labeled with both the anti-EGFRvIII monoclonal antibody and chicken anti-Myc antibody the latter being used to monitor the level of expression of the fusion protein. Following a wash step, binding of the primary antibodies is probed by a two-color indirect fluorescence labeling using a specific mouse and chicken secondary antibodies for each of the primary antibody respectively.

The anti-EGFRvIII monoclonal antibodies were binding with similar signal intensities to both full length hEGFRvIII protein and small peptides of the same protein, as well to both native and heat denatured yeast displayed antigen fragments, suggesting that the epitopes for the mAbs are contained within a continuous peptide fragment (linear).

The results presented in FIG. 3 illustrate the binding properties of the anti-EGFRvIII monoclonal antibodies to various fragments of the EGFRvIII and wild-type EGFR protein cells displayed on yeast cells.

Based on the results presented in FIG. 3, the smallest EGFRvIII fragment (peptide) that the anti-EGFRvIII antibodies were able to bind was identified (Table 2). Two different epitope bins were identified: 1) aa 1-18 recognized by 1D2 as well as the 13.1.2 control mAb, 2) aa 15-37 recognized by 5B7 and 3D12. Surprisingly, despite the fact that all antibodies were capable of binding specifically to EGFRvIII expressing cells, the 5B7 and 3D12 monoclonal antibodies bind to an epitope that doesn't encompass the spliced junction neoepitope (aa 5-7) found exclusively in EGFRvIII. Despite the absence of binding on U87MG-EGFR WT expressing cells. 5B7 did bind slightly to yeast cells engineered to express EGFR WT fragments 266-482 (FIG. 3).

TABLE 2 Summary of epitope binning Epitope Containing mAb Clone Name EGFRvIII Fragment 5B7 aa 15-37  3D12 aa 15-37  1D2 aa 1-18   hFc-13.1.2 aa 1-18   225 aa 43-456

Example 5. Fine Epitope Mapping using Yeast Surface Display

To further characterize mAb epitopes within the EGFRvIII 15-37 region, an alanine scan of this region was performed, and modified fragments were expressed at the surface of the yeast. SEQ ID NO:5 shows the amino acid sequence of the EGFRvIII 15-37 fragment, where each underlined amino acid was mutated to alanine. The resulting DNAs were expressed at the surface of the yeast and each anti-EGFRvIII mAbs was tested on the corresponding yeast mutant strain by flow cytometry analysis. The original Ala19 and Ala22 were not mutated. Thus, this assay determined the contribution of each amino acid(s) in mAb binding in comparison to the original wild-type fragment (SEQ ID NO:5) which is attributed the value of 100%.

Table 3 shows the results obtained in flow cytometry analysis for the 5B7 and 3D12 monoclonal antibodies. Results obtained are in line with the results of FIG. 3 i.e. binding of mAbs in bin 15-37 are strongly inhibited by mutation of at least one amino acid residue between position 15 and 19. This assay shows that the binding of the 5B7 monoclonal antibody is compromised by mutation of Val17, Arg18, Cys20, Gly21, Asp23 and Cys35. The binding of the 3D12 monoclonal antibody is strongly inhibited by mutation of Arg18, Cys20, Gly21, Tyr25, Glu26, Gly31 and Cys35, and weakened by Glu29 and Arg33 mutation to Alanine.

TABLE 3 Flow cytometry evaluation of mAb binding to yeast expressing mutated amino acid within fragment 15-37 of EGFRvIII. Data represent the % of binding of mAb on the yeast displaying the mutated sequence compared to the binding on the yeast displaying the wild-type sequence, normalized to the Myc-tag expression. Amino acid mutated to mAbs Corresponding Ala 5B7-2 3D12-2 peptide sequence Ser15 91.6 90.2 SEQ ID: 48 Cys16 240.2 170.7 SEQ ID: 49 Val17 0.3 116.2 SEQ ID: 50 Arg18 0.2 0.1 SEQ ID: 51 Ala19* ND ND — Cys20 0.4 0.2 SEQ ID: 52 Gly21 0.1 0.0 SEQ ID: 53 Ala22* ND ND — Asp23 0.8 62.4 SEQ ID: 54 Ser24 230.1 232.8 SEQ ID: 55 Tyr25 192.5 3.5 SEQ ID: 56 Glu26 140.1 1.4 SEQ ID: 57 Met27 111.4 75.3 SEQ ID: 58 Glu28 78.5 71.0 SEQ ID: 59 Glu29 109.7 47.7 SEQ ID: 60 Asp30 105.6 78.5 SEQ ID: 61 Gly31 98.9 10.7 SEQ ID: 62 Val32 101.1 93.8 SEQ ID: 63 Arg 33 95.8 22.1 SEQ ID: 64 Lys34 192.7 137.0 SEQ ID: 65 Cys35 1.5 0.1 SEQ ID: 66 Lys36 87.2 95.1 SEQ ID: 67 Lys37 143.3 162.3 SEQ ID: 68 *Original Ala 19 and Ala 22 were not mutated Legend Values between 0% and 15%: No binding Values between 16% and 59%: Partial binding Values at or above 60%: Complete binding

Example 6: Antibody Sequencing

The sequence of the VH and VL regions of the anti-EGFRvIII antibodies 5B7, 3D12 and 1D2 were analyzed.

Briefly, total RNA was extracted from hybridoma clones (Qiagen, RN Easy) and reverse transcribed into cDNA (SuperScript™, ThermoFisher Scientific, Waltham, Mass., USA). DNA encoding VH and VL domains was PCR amplified (Platinum Taq or equivalent) using mixtures of degenerate forward primers annealing in FR1 and a single reverse primer annealing in CH1 (Novagen/EMD Millipore cat. no 69831-3). The resulting amplicons were sequenced using the Sanger method on an ABI 3730xl instrument or were determined using 2×250 bp reads on an Illumina MiSeq instrument.

Sequences of the VH and VL regions as well as the CDR regions are shown in the Sequence Table section. Analysis of the sequence for a consensus binding sequence of the CDR 1-3 regions of the VH and VL chains was conducted using Kabat numbering scheme (Kabat et al 1992, Johnson et al 2004). The results of this analysis indicated that 5B7, 3D12 and 1D2 monoclonal antibodies have unique VH and VL CDRs.

Example 7: EGFRvIII CAR-T Design and Cloning

To generate a chimeric antigen receptor sequence using the EGFRvIII-specific antibody sequences (as described above), amino acid sequences based on the variable regions of the 5B7, 3D12, 1D2 monoclonal antibodies were assembled into single chain variable fragments (scFv) containing their respective heavy chain, a (GGGGS)3 linker sequence (although any appropriate linker could be used), followed by the light chain in silico. An amino acid sequence compatible with a restriction site was included at the beginning of the linker sequence to allow later recombination as needed. In addition, a spacer sequence was attached to the 3′ end of the sequence. In the present case, a hinge domain derived from mouse CD8 was used, but this sequence can also be substituted for other spacer domains as described herein.

FIG. 4 illustrates the synthetic assembly of 5B7 amino acids (SEQ ID NO: 30) for insertion in CAR vector to generate EGFRvIII 5B7 CAR (SEQ ID NO:32). Sequence shows the heavy chain portion (underlined), amino acids corresponding to a restriction site (*), linker (L), the light chain portion (underlined), and the mouse CD8 hinge. The 3D12 and 1 D2 sequences were assembled similarly (SEQ ID NO:33 and SEQ ID NO:35 respectively) to generate EGFRvIII 3D12 CAR (SEQ ID NO:34) and EGFRvIII 1D2 CAR (SEQ ID NO:36) respectively.

After in silico assembly, sequences were reverse translated into DNA using a standard human codon usage table via online tool available to the public (Chojnacki, S. et al., Nucl Acid Res. 45(W1): W550-553, 2017). Specific DNA sequences for various restriction sites were avoided and appropriate DNA linkers were added to the ends of the DNA to allow insertion of the sequence into a modular CAR vector using a scarless cloning strategy based on a Type IIS restriction enzyme (BbsI). DNA was then synthesized by Integrated DNA Technologies in the form of a linear double stranded gene fragment.

The DNA sequence of the 5B7 scFv and hinge as synthesized is presented in SEQ ID NO: 31. Sequence shows heavy chain (underlined), restriction sites (nucleotide 5 to 10, 412 to 418 and 1039 to 1045), linker (419 to 457), light chain (underlined), and mouse CD8 hinge (twice underlined). Sticky end sequences for scarless insertion via type Ils cloning are shown in bold.

This sequence was then inserted into our modular CAR vector (SEQ ID NO:37, FIG. 5A). The modular CAR vector comprises nucleic acid sequence encoding a human CD28 signal peptide (nucleotide 1 to 50), restriction sites (nucleotides 57 to 63 and 65 to 70) including sticky end sequences shown in black, filler (irrelevant sequence), a human CD28 transmembrane domain (nucleotide 84 to 165), a human CD28 intracellular transduction domain (nucleotide 167 to 294), and a human CD3 zeta intracellular transduction domain (nucleotide 295 to 642). Linker sequences for scarless insertion via type IIs cloning are shown in bold.

Certain truncations of the CAR sequences especially truncations in the scFv portion (as exemplified in SEQ ID NO:78) can retain similar activity while removing some backbone elements of the antibody heavy or light chain. Exemplary data is provided to show that a CAR plasmid containing truncated versions of both the VH and VL elements of 1D2-CAR has minimal effect on the ability of CAR-transduced T cells to respond to target cells as measured using electroporated Jurkat cells exposed to varying doses of EGFRvIII+target cells (FIG. 5B and FIG. 5C).

Sequences were combined into a modular plasmid vector which also contained a human EF1a promoter, P2A-GFP marker and Lentiviral transfer elements. Construction was confirmed using clonal sequencing in Escherichia coli bacteria, and plasmids were isolated by standard technique.

Alternatively, the scFv sequences described herein can be inserted into vectors for expression of bi-specific T-cell engagers, bispecific killer cell engagers or trispecific killer cell engagers (FIG. 5D).

Example 8: Detection of CAR Expression on Transduced Cells

Two strategies were employed to detect the expression of CAR molecule(s) on transduced immune cells. As a research strategy, the green fluorescence protein (GFP) was co-expressed with the EGFRvIII CAR and used as a surrogate for monitoring CAR expression in transduced cells.

Briefly, the Jurkat human CD8⁺ T-cell line was transiently transfected with the EGFRvIII construct which also expressed GFP using a 2A self-cleaving peptide based multi-gene expressing system. Expression of EGFP was quantified using flow cytometry (FIG. 6A) and the surface expression of CAR was detected by flow cytometry using a fluorescent tagged antibody that recognize murine IgG (Goat Anti-Mouse IgG H&L [Alexa Fluor® 594]; Jackson Immunoresearch) (FIG. 6B).

The flow cytometry analysis showed an increased level of GFP expression in EGFRvIII CAR (EGFRvIII 5B7 CAR (a.k.a. 5B7), EGFRvIII 3D12 CAR (a.k.a. 3D12) and EGFRvIII 1 D2 CAR (a.k.a. 1 D2)) transduced cells compared to the control cells (FIG. 6A).

In addition, an antibody recognizing murine IgG sequences within the scFv (GAMFab) was used for direct evaluation of surface expression of the CAR. The EGFRvIII CAR (PSLQC2-5B7 and 1D2) transduced cells were positive for GFP expression and binding of the GAMFab antibody whereas the control cells that were transduced with GFP expressing control plasmid devoid of a CAR construct (PX458) showed GFP expression but no binding of the GAMFab antibody (FIG. 6B).

Example 9: CAR-T Functionality Screening

The in vitro functionality of the EGFRvIII CAR constructs was tested using a novel flow cytometry based high-throughput screening platform developed by the Applicant; which is in some instances referred to as CAR-J assay. In brief, EGFRvIII or control (CD19-targeted) CAR plasmids were electroporated into the Jurkat human CD8⁺ T-cell line. Cells expressing CAR were then exposed to various target cell lines (with or without EGFRvIII expression) in varying doses and maintained under standard culture conditions. Following 24 hours of co-incubation with target cells, CAR-T cells were examined for cellular activation by flow cytometry via surface expression of the T-cell activation marker CD69. The level of auto-activation (tonic signaling) associated with each CAR was also examined by quantification of the level of CD69 expression on non-stimulated CAR-expressing Jurkat cells or CAR-expressing Jurkat cells incubated with irrelevant target cells. The high-throughput screening method for CAR functionality is summarized in FIG. 7A.

Jurkat cells electroporated with various CAR constructs (X1, X2, 5B7, 3D12 and 1D2) were exposed to varying doses of target cells with (U87vIII, DKMG) or without (U87-MG) specific target expression (FIG. 7B, C and D). Tonic signaling/auto-activation of each CAR construct was assessed by examining the level of CD69 expression in CAR-expressing (GFP+) cells without additional stimulation (FIG. 7B). The relative increase in expression of CD69 within CAR cells following stimulation with EGFRvIII-expressing target cells is illustrated in FIG. 7C and FIG. 7D.

The results of FIGS. 7B-D indicate that some CAR constructs (X1 and X2) showed high level of activation in the absence of stimulation or when stimulated with target cells that do not express EGFRvIII (U87-MG), indicative of high tonic signaling and therefore would not be suitable for CAR-mediated therapy. The 5B7, 3D12 and 1D2 CAR constructs showed very low level of activation in the absence of stimulation or in response to irrelevant target cells (U87-MG, FIG. 7B) but showed high level of activation in response to specific stimulation with EGFRvIII expressing target cells U87vIII and DKMG (FIGS. 7C and D, respectively). This allowed the selection of the 5B7, 3D12 and 1D2 CAR constructs for further characterization.

In a separate experiment, plasmid expressing CAR targeting EGFRvIII or control plasmids (PX458 expressing GFP but no CAR construct and plasmid expressing CAR targeting wild-type EGFR protein) were electroporated into Jurkat cells. GFP expressing cells and control Jurkat cells (CD45 positive) were then exposed to various target cell lines with (U87vIII) or without (A20 and U87-MG) EGFRvIII expression in varying doses and maintained under standard culture conditions. Following 24 hours of incubation, cells were examined via flow cytometry for GFP expression and cell activation using fluorescent staining against the T-cell activation marker CD69. The level of auto-activation (tonic signaling) associated with each CAR was also examined by quantification of the level of CD69 expression on non-stimulated CAR-expressing Jurkat cells (FIG. 8A).

The baseline level of CD69 expression in unstimulated CAR transduced Jurkat cells is similar to levels seen with unstimulated wild-type Jurkat cells suggesting no detectable tonic signaling in CAR-T cells. Similarly, low level of CD69 expression that were comparable to levels seen with wild-type Jurkat cells was seen with EGFR wild-type and EGFRvIII CAR-T cells when stimulated with human B cell lymphoma cell line A20 that does not express wild-type EGFR or EGFRvIII protein. CAR-T cells targeting wild-type EGFR responded strongly to U87, U87vIII and OVCAR3 cells, which express the wild-type EGFR protein whereas EGFRvIII targeted CAR-T cells responded only to U87vIII cells and not the U87 or OVCAR3 cells (FIG. 8A).

In order to optimize the length of the hinge element that should be integrated in CAR constructs, various constructs were tested using Jurkat activation assay similarly as described above. Briefly, CAR constructs containing either 5B7 scFV (SEQ ID NO:44) or 3D12 scFV (SEQ ID NO:45) sequence followed by (1) a very long hinge wherein a 17AA poly-glycine linker [(GGGGS)₄GG] was followed by the 45AA sequence of the hinge domain of the human CD8 protein [L17-CD8h: SEQ ID NO:83], (2) human CD8 hinge alone [45CD8h: SEQ ID NO:84], (3) a truncated form of CD8-hinge containing C-terminal 35AA [35CD8h: SEQ ID NO:85], (4) a truncated form of CD8-hinge containing C-terminal 15AA [15CD8h: SEQ ID NO:86], or (5) a truncated form of CD8-hinge containing C-terminal 1AA [1CD8h: SEQ ID NO:87] were generated. Jurkat cells were then electroporated with vlll-specific constructs with varying hinge length and co-cultured at a 1:1 effector to target ratio with antigen expressing cells (U87III) or antigen-negative cells (U87WT). Results clearly demonstrate that EGFRvIII constructs developed here show high target-specific response across a range of hinge lengths (FIG. 8B).

Based on the in vitro functionality data, EGFRvIII CAR constructs 1D2 and 3D12 were selected for testing for in vivo functionality.

Briefly, athymic nude mice (Jackson Laboratory, Barr Harbor, Me.) were injected subcutaneously with 1'10⁶ (FIG. 9A) or 4×10⁶ (FIG. 9B) U87vIII human glioblastoma cells expressing EGFRvIII. CD19 targeted CAR-T was used as control in the studies. On day 5 post tumor cell injection, mice were given 10⁵ (FIG. 9A) or 10⁶ (FIG. 9B) human primary T cells expressing EGFRvIII 1D2 CAR. Animals were monitored for tumor growth. Tumor size (the length and the width) was measured using a digital vernier caliper. Tumor volume was calculated by using the formula: Tumor volume=(0.4) (ab2), where a=large diameter and b=smaller diameter. The tumor volume (mean±SEM) is depicted in FIG. 9A and FIG. 9B. FIG. 9C depicts the level of CD8⁺ T-cell infiltration in tumors from two animals each from groups receiving EGFRvIII targeted CAR-T and showing good tumor control and irrelevant CAR (CD19 CAR-T).

The results illustrate an increase in tumor growth in all animals receiving irrelevant CD19 CAR-T cells whereas effective tumor control was seen in a subset of animals receiving EGFRvIII CAR-T cells (FIGS. 9A and FIG. 9B). EGFRvIII CAR-T treated mice showing good tumor control also showed good CAR-T cell infiltration in the tumor (FIGS. 9C).

Example 10: CAR-NK Functional Screening

The human NK-92 cell line (Gong et al; 1994; Leukemia) was used for development and testing of EGFRvIII targeted NK 3D12-CAR cells. The graphic in FIG. 10A represents the generation of EGFRvIII targeted NK92-CAR cells for in vitro testing of tumor cytotoxicity. In brief, EGFRvIII or control (wild-type EGFR protein-targeted) CAR plasmids were electroporated into NK-92 cells (ATCC; Manassas, Va.). Cells expressing CAR were then exposed to various target cell lines with (U87vIII) or without (U87) EGFRvIII expression in varying doses and maintained under standard culture conditions. Following 24 hours of co-incubation with target cells, tumor cell killing was assessed by quantifying the release of lactate dehydrogenase (FIG. 10B LDH; a cytosolic enzyme which is release upon cell death) by colorimetry or by using the standard ⁵¹Cr-relase assay (FIG. 10C). Wild-type NK92 cells devoid of CAR construct was used as negative control.

EGFR WT NK-CAR showed killing of U87 and U87vIII cells both of which overexpress a common epitope present on both EGFR protein isoforms whereas EGFRvIII CAR-NK showed preferential killing of U87vIII cells. The specific cytotoxicity of EGFRvIII targeted NK 3D12-CAR towards EGFRvIII and not the wild-type EGFR protein expressing cells was shown by both LDH (FIG. 10B) and ⁵¹Chromium release (FIG. 10C) assays.

The in vivo functionality of the EGFRvIII targeted NK-CARs was also tested using 2 independent experiments using athymic nude mice bearing EGFRvIII expressing U87vIII tumors.

Briefly, athymic nude mice (Jackson Laboratory, Barr Harbor, Me.) were injected subcutaneously with 1'10⁶ U87vIII human glioblastoma cells expressing EGFRvIII. On day 2 post tumor cell injection, mice were given either 5×10⁶ wild-type NK92 cells devoid of CAR or NK92 cells expressing EGFRvIII 3D12-CAR-NK (FIG. 11A) or on day 3 post tumor induction animals were given 1×10⁷ EGFRvIII 3D12-CAR-NK or left untreated (FIG. 11B). Animals were monitored for survival (left panels) and tumor growth (right panels). Tumor size (the length and the width) was measured using a digital vernier caliper. Tumor volume was calculated by using the formula: Tumor volume=(0.4) (ab2), where a=large diameter and b=smaller diameter.

In both studies, better control of tumor growth and increased survival was seen in animals receiving EGFRvIII NK-CAR compared to animals receiving unmodified NK92 cells or untreated controls (FIG. 11A and FIG. 11B) respectively.

The chimeric antigen receptors (CAR) generated herein may therefore be used to re-direct NK cells to specifically recognize and kill cells expressing EGFRvIII protein.

Example 11: Primary CAR-T Functional Testing In Vitro

Human primary peripheral blood derived T cells were used for confirmation of in vitro and in vivo activity of EGFRvIII targeted 3D12 CARs constructs in the context of primary human immune cells. In brief, EGFRvIII-CAR or control (human CD19-targeted FMC63-CAR) lentivirus was generated using standard production protocols in HEK293 and concentrated using ultracentrifugation. Primary T cells were isolated from donor blood samples using magnetic separation and activated using anti-CD3/CD28 beads. Primary T cells were then transduced with CAR lentivirus and expanded for several days in culture.

In vitro functionality of primary human EGFRvIII-specific CAR-transduced T cells were assessed using a live-fluorescence microscopy approach. Briefly, EGFRvIII targeted CAR-T or mock transduced T cells, wherein no lentiviral construct was introduced into cells handled under similar conditions, were generated as described above. Cells were then placed in co-culture with EGFRvIII-expressing target cells modified to also express a nuclear-localized form of mKate2 fluorescent protein. Co-cultures were monitored constantly over 6 days using the IncucyteTM automated live fluorescent microscopy device (Sartorius, USA). The relative growth of target cells was then assessed using automated counting of mKate2+ cells (FIG. 12). Data showed that the growth of U87MG overexpressing EGFRvIII cells was efficiently repressed by 3D12 CAR-T compared to mock transfected T-cells.

Example 12: Primary CAR-T Functional Testing In Vivo

The in vivo functionality of the EGFRvIII targeted primary CAR-T cells was also tested using Nod-SCID-IL2γR2^(−/−)(NSG) mice (Jackson Laboratory, Barr Harbor, Me.) bearing EGFRvIII expressing U87vIII tumors. Briefly, mice were subcutaneously injected with 1×10⁶ fluorescently labelled U87-vIII cells. Eight days after tumour cell injection, cryo-preserved CAR-T cells were thawed, washed with PBS, and 1×10⁷ total T cells (with 20-25% CAR transduction) were immediately delivered intra-tumourally, ensuring equal distribution of tumour sizes between groups. Tumour growth was evaluated three times per week using calipers by trained animal technicians blinded to specific treatment groups (FIG. 13A). Primary endpoint was tumour size above 2000 mm³, with secondary endpoints determined by overall animal health and well-being (FIG. 13B). Tumor volume was calculated by using the formula: Tumor volume=(0.4) (ab2), where a=large diameter and b=smaller diameter.

In this study, better control of tumor growth and increased survival was seen in animals receiving EGFRvIII primary CAR-T compared to untreated control animals (FIG. 13A and FIG. 13B) respectively.

Example 13: Bi-specific Immune Cell Engager Functional Testing

Various experiments were performed to demonstrate the activity of the novel EGFRvIII-specific single chain variable fragment in the context of a bispecific T cell engager. Constructs were generated using synthetic DNA wherein the 3D12 scFV sequence (SEQ ID NO:45) was linked to a previously demonstrated CD3-engaging scFv sequence (SEQ ID NO:82). A plasmid expressing this bi-specific construct was transfected into human embryonic kidney cells (HEK293T) and supernatant was collected after 2 to 4 days in culture. Supernatant from HEK293T were transferred to wells containing Jurkat cells and varying doses of antigen expressing target cells (U87vIII). Target-induced activation in the presence or absence of bispecific T-cell engager was measured by examining the level of CD69 expression using human CD69-specific antibody staining and flow cytometry (FIG. 14). Results showed that only EGFRvIII targeted bi-specific construct could induce high target-specific T-cell activation response compared to a control bi-specific molecule.

In vitro functionality of primary human EGFRvIII-specific bi-specific immune T-cell engager in interaction with primary human T cells were assessed using a live-fluorescence microscopy approach. Briefly, polyclonally expanded human T cells which were allowed to return to a rest state over several weeks in culture were placed in co-culture with EGFRvIII-expressing target cells modified to also express a nuclear-localized form of mKate2 fluorescent protein. Various doses of HEK293T supernatant, or supernatant wherein cells were secreting a control CD19-CD3 targeted or 3D12-CD3 targeted bi-specific immune cell engager were transferred to T-cell target cell co-cultures. Co-cultures were then monitored constantly over 6 days using the IncucyteTM automated live fluorescent microscopy device (Sartorius, USA). The relative growth of target cells was then assessed using automated counting of mKate2+ cells (FIG. 15). Results demonstrate that 3D12-CD3 bi-specific immune cell engagers can actively induce T-cell mediated repression of EGFRvIII-positive tumour cell growth compared to the bi-specific CD19-CD3 control molecule or mock supernatant.

The embodiments and examples described herein are illustrative and are not meant to limit the scope of the disclosure as claimed. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventors to be encompassed by the claims. Citations listed in the present application are incorporated herein by reference.

REFERENCES

All patents, patent applications and publications referred to throughout the application are incorporated herein by reference.

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SEQUENCE TABLE CDRs are generally indicated in bold and/or underlined Seq. ID Description Sequence  1 Wild type human EGFR CTGGAGGAAAAGAAAGTTTGCCAAGGCACGAGTAACAAGCTCACGCAGTT ectodomain cDNA sequence GGGCACTTTTGAAGATCATTTTCTCAGCCTCCAGAGGATGTTCAATAACT GTGAGGTGGTCCTTGGGAATTTGGAAATTACCTATGTGCAGAGGAATTAT GATCTTTCCTTCTTAAAGACCATCCAGGAGGTGGCTGGTTATGTCCTCAT TGCCCTCAACACAGTGGAGCGAATTCCTTTGGAAAACCTGCAGATCATCA GAGGAAATATGTACTACGAAAATTCCTATGCCTTAGCAGTCTTATCTAAC TATGATGCAAATAAAACCGGACTGAAGGAGCTGCCCATGAGAAATTTACA GGAAATCCTGCATGGCGCCGTGCGGTTCAGCAACAACCCTGCCCTGTGCA ACGTGGAGAGCATCCAGTGGCGGGACATAGTCAGCAGTGACTTTCTCAGC AACATGTCGATGGACTTCCAGAACCACCTGGGCAGCTGCCAAAAGTGTGA TCCAAGCTGTCCCAATGGGAGCTGCTGGGGTGCAGGAGAGGAGAACTGCC AGAAACTGACCAAAATCATCTGTGCCCAGCAGTGCTCCGGGCGCTGCCGT GGCAAGTCCCCCAGTGACTGCTGCCACAACCAGTGTGCTGCAGGCTGCAC AGGCCCCCGGGAGAGCGACTGCCTGGTCTGCCGCAAATTCCGAGACGAAG CCACGTGCAAGGACACCTGCCCCCCACTCATGCTCTACAACCCCACCACG TACCAGATGGATGTGAACCCCGAGGGCAAATACAGCTTTGGTGCCACCTG CGTGAAGAAGTGTCCCCGTAATTATGTGGTGACAGATCACGGCTCGTGCG TCCGAGCCTGTGGGGCCGACAGCTATGAGATGGAGGAAGACGGCGTCCGC AAGTGTAAGAAGTGCGAAGGGCCTTGCCGCAAAGTGTGTAACGGAATAGG TATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAAC ACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTG GCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGA ACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTC AGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAA ATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGT CAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTG ATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACA ATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTAT AAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATG CCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTC TCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCT TCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGT GCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGG GGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTG CGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCT GGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGC ACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCC TAAGATCCCGTCC  2 Human EGFR ectodomain LEEKKVCQGTSNKLTQLGTFEDHELSLQRMENNCEVVLGNLEITYVQRNY amino acid sequence DLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSN YDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLS NMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCR GKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTT YQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVR KCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPV AFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLE IIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANT INWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCV SCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGR GPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNC TYGCTGPGLEGCPTNGPKIPS  3 Human EGFRvIII ectodomain CTGGAAGAGAAGAAAGGCAACTACGTCGTGACCGACCACGGCAGCTGTGT cDNA GCGGGCTTGTGGCGCCGATAGCTACGAGATGGAAGAGGACGGCGTGCGGA sequence AGTGCAAGAAGTGCGAGGGCCCCTGCCGGAAAGTGTGCAACGGCATCGGC (nucleotides 1-996) ATCGGAGAGTTCAAGGACAGCCTGAGCATCAACGCCACCAACATCAAGCA CTTCAAGAACTGCACCAGCATCAGCGGCGACCTGCACATCCTGCCCGTGG CCTTTAGAGGCGACAGCTTCACCCACACCCCCCCACTGGACCCCCAGGAA CTGGACATCCTGAAAACCGTGAAAGAGATCACCGGCTTTCTGCTGATTCA GGCCTGGCCCGAGAACCGGACAGACCTGCACGCCTTCGAGAACCTGGAAA TCATCCGGGGCAGGACCAAGCAGCACGGCCAGTTTTCTCTGGCCGTGGTG TCCCTGAACATCACCAGCCTGGGCCTGCGGAGCCTGAAAGAAATCAGCGA CGGCGACGTGATCATCTCCGGCAACAAGAACCTGTGCTACGCCAACACCA TCAACTGGAAGAAGCTGTTCGGCACCTCCGGCCAGAAAACAAAGATCATC AGCAACCGGGGCGAGAACAGCTGCAAGGCCACAGGACAAGTGTGCCACGC CCTGTGTAGCCCTGAGGGCTGTTGGGGACCCGAGCCCAGAGATTGCGTGT CCTGCAGAAACGTGTCCCGGGGCAGAGAATGCGTGGACAAGTGCAACCTG CTGGAAGGCGAGCCCCGCGAGTTCGTGGAAAACAGCGAGTGCATCCAGTG CCACCCCGAGTGTCTGCCCCAGGCCATGAACATTACCTGCACCGGCAGAG GCCCCGACAACTGTATCCAGTGCGCCCACTACATCGACGGCCCCCACTGC GTGAAAACCTGTCCTGCTGGCGTGATGGGAGAGAACAACACCCTCGTGTG GAAGTACGCCGACGCCGGCCATGTGTGCCACCTGTGTCACCCCAAT  4 Human EGFRvIII ectodomain LEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIG amino acid sequence IGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQE (amino acids 1-332) LDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVV SLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKII SNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNL LEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHC VKTCPAGVMGENNTLVWKYADAGHVCHLCHPN  5 Human SCVRACGADSYEMEEDGVRKCKK EGFRvIII amino acid residues 15 to 37  6 5B7 light chain variable region DVVMTQTPLTLSVTIGQPASISC

WLLQRPGQSPK (CDRs in bold) RLIY

GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC

FGGGTKLEIK  7 5B7 CDRL1 KSSQSLLDSDGKTYLN  8 5B7 CDRL2 LVSKLDS  9 5B7 CDRL3 WQGTHFPQT 10 5B7 heavy QIQLVQSGPELKKPGETVMISCKASGYSFT

WVKQAPEKDLKWMG

chain variable

RFAFSLETSVSIVYLKINNLKNEDMATYFCAR

region

WGTGTTVTVSS 11 5B7 CDRH1 NYGMN 12 5B7 CDRH2 WINTYTGESRYVDEFKG 13 5B7 CDRH3 GPNFDV 14 3D12 light EIVLTQSPALMAASPGEKVTITC

WYQQKSETSPKPWIY chain variable

GVPVRFSGSGSGTSYSLTISSMEAEDAATYYC

FG region SGTKLEIK 15 3D12 CDRL1 SVSSSISSSNLH 16 3D12 CDRL2 GTSNLAS 17 3D12 CDRL3 QQWSSYPLT 18 3D12 heavy EVQLQQSGPELVKPGSSVRISCKASGYTFT

WVKQSHGKSLEWIG

chain variable

KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR

region

WGQGTLVTVSA 19 3D12 CDRH1 DYNMD 20 3D12 CDRH2 TINPNNGGTSYNQKFKG 21 3D12 CDRH3 VRQLGLWFAY 22 1D2 light chain DVVMTQTPLSLPVSLGDQASISC

WYLQKPGQSPK variable region LLIY

GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC

FGGGTKLEIK 23 1D2 CDRL1 RSSQSLVHSDGNTYLH 24 1D2 CDRL2 KVSNRFS 25 1D2 CDRL3 SQSTHVPWT 26 1D2 heavy DVQLQESGPDLVKPSQSLSLTCTVTGYSIT

WIRQFPGNKLEWMG chain variable

RISITRDTSKNQFFLQLSSVTTEDTATYYCTR

region

WGTGTTVTVSSA 27 1D2 CDRH1 SGYSWH 28 1D2 CDRH2 YIHYSGSTNYNPSLKS 29 1D2 CDRH3 SPYFDV 30 5B7 CAR QIQLVQSGPELKKPGETVMISCKASGYSFT

WVKQAPEKDLKWMG

construct amino acid sequence

RFAFSLETSVSIVYLKINNLKNEDMATYFCAR

(with linker and CD8 hinge)

WGTGTTVTVSSAKTTAPSVYPLAPGSLGGTGGGSGGGGSGGGGSDV VMTQTPLTLSVTIGQPASISC

WLLQRPGQSPKRL IY

GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC

FGGGTKLEIKRADAAPTVSIFPPSSKLGVISNSVMYFSSVVPVLQKVNST TTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFA 31 5B7 CAR construct GACAGAAGACCTAGGACAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGA nucleotide sequence AGAAGCCTGGAGAGACAGTCATGATCTCCTGCAAGGCTTCTGGGTATTCC TTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGAAAAGGATTT AAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGTCAAGATATGTTG ATGAATTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGTTAGTATT GTCTATTTGAAGATCAACAACCTCAAAAATGAGGACATGGCTACATATTT CTGTGCAAGAGGGCCTAATTTCGATGTCTGGGGCACAGGGACCACGGTCA CTGTCTCCTCAGCCAAAACAACAGCCCCATCCGTCTATCCCCTGGCCCCT GGAAGCTTGGGAGGTACCGGCGGAAGTGGAGGCGGAGGATCTGGCGGCGG AGGATCCGATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCA TTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGAT AGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTC TCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTG ACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGC AGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACA TTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTG ATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAAGCTTGGGGTC ATCAGCAACTCGGTGATGTACTTCAGTTCTGTCGTGCCAGTCCTTCAGAA AGTGAACTCTACTACTACCAAGCCAGTGCTGCGAACTCCCTCACCTGTGC ACCCTACCGGGACATCTCAGCCCCAAAGACCAGAAGATTGTCGGCCCCGT GGCTCAGTGAAGGGGACCGGATTGGACTTCGCCCCTTGGGTCTTCGGTAG GG 32 5B7 CAR MLRLLLALNLFPSIQVTGQTQLVQSGPELKKPGETVMISCKASGYSET

construct with CD28

WVKQAPEKDLKWMG

RFAFSLETSVSIVYLK intracellular transduction INNLKNEDMATYFCAR

WGTGTTVTVSSAKTTAPSVYPLAPGSLG domain (ITD) and CD3zeta GTGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISC

ITD (signal peptide underlined)

WLLQRPGQSPKRLIY

GVPDRFTGSGSGTDFTLKISRVE AEDLGVYYC

FGGGTKLEIKRADAAPTVSIFPPSSKLGVISN SVMYFSSVVPVLQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSV KGTGLDFAPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSD YMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASLRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE 33 3D12 CAR construct amino EVQLQQSGPELVKPGSSVRISCKASGYTFT

WVKQSHGKSLEWIG

acid sequence

KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR

(with linker and CD8 hinge)

WGQGTLVTVSAAKTTPPSVYPLAPGSLGGTGGGSGGGGSGGG GSEIVLTQSPALMAASPGEKVTITC

WYQQKSETSPKPW IY

GVPVRFSGSGSGTSYSLTISSMEAEDAATYYC

FGSGTKLEIKRADAAPTVSIFPPSSKLGVISNSVMYESSVVPVLQKVNST TTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFA 34 3D12 CAR construct with MLRLLLALNLFPSIQVTGEVQLQQSGPELVKPGSSVRISCKASGYTFT

CD28 ITD and CD3zeta ITD

WVKQSHGKSLEWIG

KATLTVDKSSSTAYME (signal peptide underlined) LRSLTSEDSAVYYCAR

WGQGTLVTVSAAKTTPPSVYPLAP GSLGGTGGGSGGGGSGGGGSEIVLTQSPALMAASPGEKVTITC

WYQQKSETSPKPWIY

GVPVRFSGSGSGTSYSLTISSME AEDAATYYC

FGSGTKLEIKRADAAPTVSIFPPSSKLGVISN SVMYFSSVVPVLQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSV KGTGLDFAPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSD YMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASLRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE 35 1D2 CAR DVQLQESGPDLVKPSQSLSLTCTVTGYSIT

WIRQFPGNKLEWMG construct amino acid sequence

RISITRDTSKKEELQLSSVTTEDTATYYCTR

(with linker and CD8 hinge)

WGTGTTVTVSSAKTTPPSVYPLAPGSLGTGGGSGGGGSGGGGSDVV MTQTPLSLPVSLGDQASISC

WYLQKPGQSPKLLI Y

GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC

F GGGTKLEIKRADAAPTVSIFPPSSKLGVISNSVMYFSSVVPVLQKVNSTT TKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFA 36 1D2 CAR MLRLLLALNLFPSIQVTGDVQLQESGPDLVKPSQSLSLTCTVTGYSIT

construct with CD28 ITD and

WIRQFPGNKLEWMG

RISITRDTSKNQFFLQ CD3zeta ITD LSSVTTEDTATYYCTR

WGTGTTVTVSSAKTTPPSVYPLAPGSLG (signal peptide underlined) TGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISC

WYLQKPGQSPKLLIY

GVPDRFSGSGSGTDFTLKISRVEA EDLGVYFC

FGGGTKLEIKRADAAPTVSIFPPSSKLGVISNS VMYFSSVVPVLQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVK GTGLDFAPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDY MNMTPRRPGPTRKHYQPYAPPRDFAAYRSASLRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE 37 DNA sequence for modular ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTTCAATTCAAGTAAC CAR vector AGGAGGGTCTTCGAGAAGACCTCCTTCTAAGCCCTTTTGGGTGCTGGTGG (without filler/insert) TGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTT ATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTA CATGAACATGACTCCCAGGCGGCCCGGACCCACCCGCAAGCATTACCAGC CCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGCTAGCCTGAGA GTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGA AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA AGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCTCGAG 38 5B7 Heavy chain variable CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGAC region nucleotide sequence AGTCATGATCTCCTGCAAGGCTTCTGGGTATTCCTTCACAAACTATGGAA TGAACTGGGTGAAGCAGGCTCCAGAAAAGGATTTAAAGTGGATGGGCTGG ATAAACACCTACACTGGAGAGTCAAGATATGTTGATGAATTCAAGGGACG GTTTGCCTTCTCTTTGGAAACCTCTGTTAGTATTGTCTATTTGAAGATCA ACAACCTCAAAAATGAGGACATGGCTACATATTTCTGTGCAAGAGGGCCT AATTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA 39 5B7 Light chain variable region GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACA nucleotide sequence ACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATG GAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAG CGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTT CACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGG AGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCT CAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 40 3D12 Heavy chain variable GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGG

TCTTC region nucleotide sequence AGTGAGGATATCCTGCAAAGCTTCTGGATACACATTCACTGACTACAACA TGGACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAACT ATTAATCCTAACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAA GGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAACTCC GCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGTGAGA CAGCTCGGGCTGTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGT CTCTGCA 41 3D12 Light chain variable GAAATTGTGCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGGGGA region nucleotide sequence GAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCAACT TGCACTGGTA

CAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTAT GGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGG ATCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATG CTGCCACTTATTACTGTCAACAGTGGAGTAGTTACCCACTCACGTTCGGC TCGGGGACAAAGTTGGAAATAAAA 42 1D2 Heavy chain variable GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTC nucleotide sequence ACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTTATA GCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGC TACATACACTACAGTGGTAGCACTAACTACAACCCATCTCTCAAAAGTCG AATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGA GTTCTGTGACTACTGAGGACACTGCCACATATTACTGTACAAGAAGCCCG TACTTCGATGTCTGGGGCACAGGGACCACGGTCAC

GTCTCCTCA 43 1D2 Light chain variable GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGCGA region nucleotide sequence TCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTGCACAGTGATG GAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAG CTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTT CAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGG AGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCG TGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 44 5B7 scFV QTQLVQSGPELKKPGETVMISCKASGYSFT

WVKQAPEKDLKWMG

(linker twice-underlined, any

RFAFSLETSVSIVYLKINNLKNEDMATYFCAR

suitable known linker may be

WGTGTTVTVSSAKTTAPSVYPLAPGSLGGTGGGSGGGGSGGGG SDV used) VMTQTPLTLSVTIGQPASISC

WLLQRPGQSPKRL IY

GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC

FGGGTKLEIKRADAAPTVSIFPPSSKLG 45 3D12 scFV EVQLQQSGPELVKPGSSVRISCKASGYTFT

WVKQSHGKSLEWIG

(linker twice-underlined, any

KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR

suitable known linker may be

WGQGTLVTVSAAKTTPPSVYPLAPGSLGGTGGGSGGGGSGGG used) GSEIVLTQSPALMAASPGEKVTITC

WYQQKSETSPKPW IY

GVPVRFSGSGSGTSYSLTISSMEAEDAATYYC

FGSGTKLEIKRADAAPTVSIFPPSSKLG 46 1D2 scFV DVQLQESGPDLVKPSQSLSLTCTVTGYSIT

WIRQFPGNKLEWMG (linker twice-underlined, any

RISITRDTSKNQFFLQLSSVTTEDTATYYCTR

suitable known linker may be

WGTGTTVTVSSAKTTPPSVYPLAPGSLGTGGGSGGGGSGGGGSDVV used) MTQTPLSLPVSLGDQASISC

WYLQKPGQSPKLLI Y

GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC

F GGGTKLEIKRADAAPTVSIFPPSSKLG 47 Linker GGGSGGGGSGGGGS (exemplary linker, any suitable linker may be used) 48 Amino acid residues 15 to 37 of ACVRACGADSYEMEEDGVRKCKK EGFRvIII, with Ser15 to Ala mutation 49 Amino acid residues 15 to 37 of SAVRACGADSYEMEEDGVRKCKK EGFRvIII, with Cys16 to Ala mutation 50 Amino acid residues 15 to 37 of SCARACGADSYEMEEDGVRKCKK EGFRvIII, with Val17 to Ala mutation 51 Amino acid residues 15 to 37 of SCVAACGADSYEMEEDGVRKCKK EGFRvIII, with Arg18 to Ala mutation 52 Amino acid residues 15 to 37 of SCVRAAGADSYEMEEDGVRKCKK EGFRvIII, with Cys20 to Ala mutation 53 Amino acid residues 15 to 37 of SCVRACAADSYEMEEDGVRKCKK EGFRvIII, with Gly21 to Ala mutation 54 Amino acid residues 15 to 37 of SCVRACGAASYEMEEDGVRKCKK EGFRvIII, with Asp23 to Ala mutation 55 Amino acid residues 15 to 37 of SCVRACGADAYEMEEDGVRKCKK EGFRvIII, with Ser24 to Ala mutation 56 Amino acid residues 15 to 37 of SCVRACGADSAEMEEDGVRKCKK EGFRvIII, with Tyr25 to Ala mutation 57 Amino acid residues 15 to 37 of SCVRACGADSYAMEEDGVRKCKK EGFRvIII, with Glu26 to Ala mutation 58 Amino acid residues 15 to 37 of SCVRACGADSYEAEEDGVRKCKK EGFRvIII, with Met27 to Ala mutation 59 Amino acid residues 15 to 37 of SCVRACGADSYEMAEDGVRKCKK EGFRvIII, with Glu28 to Ala mutation 60 Amino acid residues 15 to 37 of SCVRACGADSYEMEADGVRKCKK EGFRvIII, with Glu29 to Ala mutation 61 Amino acid residues 15 to 37 of SCVRACGADSYEMEEAGVRKCKK EGFRvIII, with Asp30 to Ala mutation 62 Amino acid residues 15 to 37 of SCVRACGADSYEMEEDAVRKCKK EGFRvIII, with Gly31 to Ala mutation 63 Amino acid residues 15 to 37 of SCVRACGADSYEMEEDGARKCKK EGFRvIII, with Val32 to Ala mutation 64 Amino acid residues 15 to 37 of SCVRACGADSYEMEEDGVAKCKK EGFRvIII, with Arg33 to Ala mutation 65 Amino acid residues 15 to 37 of SCVRACGADSYEMEEDGVRACKK EGFRvIII, with Lys34 to Ala mutation 66 Amino acid residues 15 to 37 of SCVRACGADSYEMEEDGVRKAKK EGFRvIII, with Cys35 to Ala mutation 67 Amino acid residues 15 to 37 of SCVRACGADSYEMEEDGVRKCAK EGFRvIII, with Lys36 to Ala mutation 68 Amino acid residues 15 to 37 of SCVRACGADSYEMEEDGVRKCKA EGFRvIII, with Lys37 to Ala mutation 69 Amino acid residues 1 to 76 of LEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIG EGFRvIII IGEFKDSLSINATNIKHFKNCTSISG 70 Amino acid residues 3 to 37 of EKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKK EGFRvIII 71 Amino acid residues 1 to 18 of LEEKKGNYVVTDHGSCVR EGFRvIII 72 5B7 QIQLVQSGPELKKPGETVMISCKASGYSFT

WVKQAPEKDLKWMG

Shorter scFv

RFAFSLETSVSIVYLKINNLKNEDMATYFCAR

(linker twice-underlined, any

WGTGTTVTVSSGTGGGSGGGGSGGGG SDVVMTQTPLTLSVTIGQPA suitable known linker may be SISC

WLLQRPGQSPKRLIY

GVPDRFTG used) SGSGTDFTLKISRVEAEDLGVYYC

FGGGTKLEIK 73 5B scFv QIQLVQSGPELKKPGETVMISCKASGYSFT

WVKQAPEKDLKWMG

consensus

RFAFSLETSVSIVYLKINNLKNEDMATYFCAR

(linker twice-underlined, any

WGTGTTVTVSS[ Linker ]DVVMTQTPLTLSVTIGQPASISC

suitable known linker may be

WLLQRPGQSPKRLIY

GVPDRFTGSGSGTDFTL used) KISRVEAEDLGVYYC

FGGGTKLEI 74 5B7 CAR QIQLVQSGPELKKPGETVMISCKASGYSFT

WVKQAPEKDLKWMG

consensus

RFAFSLETSVSIVYLKINNLKNEDMATYFCAR

(linker twice-underlined, any

WGTGTTVTVSS[ Linker ]DVVMTQTPLTLSVTIGQPASISC

suitable known linker may be

WLLQRPGQSPKRLIY

GVPDRFTGSGSGTDFTL used) KISRVEAEDLGVYYC

FGGGTKLEIVISNSVMYFSSVVPVLQ KVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFAPSKPF WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTR KHYQPYAPPRDFAAYRSASLRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE 75 3D12 EVQLQQSGPELVKPGSSVRISCKASGYTFT

WVKQSHGKSLEWIG

Shorter scFV

KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR

(linker twice-underlined, any

WGQGTLVTVSAGTGGGSGGGGSGGGGSEIVLTQSPALMAASP suitable known linker may be GEKVTITC

WYQQKSETSPKPWIY

GVPVRFSG used) SGSGTSYSLTISSMEAEDAATYYC

FGSGTKLEIK 76 3D12 EVQLQQSGPELVKPGSSVRISCKASGYTFT

WVKQSHGKSLEWIG

scFV

KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR

consensus

WGQGTLVTVSA[ Linker ]EIVLTQSPALMAASPGEKVTITC

(linker twice-underlined, any

WYQQKSETSPKPWIY

GVPVRFSGSGSGTSYSL suitable known linker may be TISSMEAEDAATYYC

FGSGTKLEIK used) 77 3D12 EVQLQQSGPELVKPGSSVRISCKASGYTFT

WVKQSHGKSLEWIG

CAR consensus

KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR

(linker twice-underlined, any

WGQGTLVTVSA[ Linker ]EIVLTQSPALMAASPGEKVTITC

suitable known linker may be

WYQQKSETSPKPWIY

GVPVRFSGSGSGTSYSL used) TISSMEAEDAATYYC

FGSGTKLEIKVISNSVMYFSSVVPVL QKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFAPSKP FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRSASLRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE 78 1D2 DVQLQESGPDLVKPSQSLSLTCTVTGYSIT

WIRQFPGNKLEWMG (linker twice-underlined, any

RISITRDTSKNQFFLQLSSVTTEDTATYYCTR

suitable known linker may be

WGTGTTVTVSSAGTGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQ used) ASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLIY

GVPDRFS GSGSGTDFTLKISRVEAEDLGVYFCS

FGGGTKLEIK 79 1D2 DVQLQESGPDLVKPSQSLSLTCTVTGYSIT

WIRQFPGNKLEWMG Consensus scFV

RISITRDTSKNQFFLQLSSVTTEDTATYYCTR

(linker twice-underlined, any

WGTGTTVTVSSA[ Linker ]DVVMTQTPLSLPVSLGDQASISCRSSQ suitable known linker may be SLVHSDGNTYLHWYLQKPGQSPKLLIY

GVPDRFSGSGSGTDFT used) LKISRVEAEDLGVYFCS

FGGGTKLEIK 80 1D2 DVQLQESGPDLVKPSQSLSLTCTVTGYSIT

WIRQFPGNKLEWMG CAR Consensus

RISITRDTSKNQFFLQLSSVTTEDTATYYCTR

(linker twice-underlined, any

WGTGTTVTVSSA[ Linker ]DVVMTQTPLSLPVSLGDQASISCRSSQ suitable known linker may be SLVHSDGNTYLHWYLQKPGQSPKLLIY

GVPDRFSGSGSGTDFT used) LKISRVEAEDLGVYFCS

FGGGTKLEIKVISNSVMYFSSVVPV LQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFAPSK PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGP TRKHYQPYAPPRDFAAYRSASLRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE 81 3GGGGS-flexible linker GGGGSGGGGSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV (exemplary)and human CD8 hinge HTRGLDFACD 82 Amino acid sequence for EVQLQQSGPELVKPGSSVRISCKASGYTFTDYNMDWVKQSHGKSLEWIGT 3D12 bi-specific T cell INPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARVR engager exemplary sequence QLGLWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSLGGTGGGSGGGGSGGG Linker sequences underlined, GSEIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPW any suitable known linker may IYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLT be used FGSGTKLEIKRADAAPTVSIFPPSSKLGDLGGGGSRDD DIKLQQSGAELA CD3-specific scFv engager RPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQ shown in bold KFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQG TTLTVSSVE

VDDIQLTQSPAIMSASPGEKVTMTCRA SSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTIS SMEAEDAATYYCQQWSSNPLTFGAGTKLELK 83 Linker of the L17-CD8 PLGGGGSGGGGSGGGGSGGTTTPAPRPPTPAPTIASQPLSLRPEACRPAA construct GGAVHTRGLDFACD Restriction site scar underlined 84 Linker of the 45CD8h PLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD construct Restriction site scar underlined 85 Linker of the 35CD8h PLPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD construct Restriction site scar underlined 86 Linker of the 15CD8h PLAGGAVHTRGLDFACD construct Restriction site scar underlined 87 Linker of the 1CD8h PLD construct Restriction scar underlined 

1. An antigen-binding agent comprising an antigen-binding domain of an antibody that specifically binds to epidermal growth factor receptor variant III (EGFRvIII), wherein the antigen-binding domain comprises: a. a CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:15, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:16, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:17, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:19, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:20 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:21; b. a CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:7, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:8, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:9, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:11, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:12 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:13, or c. a CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:23, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:24, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:25, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:27, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:28 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:29.
 2. The antigen-binding agent of claim 1, wherein the antigen-binding domain comprises human or humanized framework amino acid sequences.
 3. The antigen-binding agent of claim 1, wherein the antigen-binding agent is an antibody, an antigen-binding fragment thereof, a chimeric antigen receptor, a bi-specific T-cell engager, a bispecific killer cell engager, a trispecific killer cell engager or any immunotherapeutic compound.
 4. (canceled)
 5. The antigen-binding agent of claim 3, wherein the antigen-binding agent is an antibody or an antigen-binding fragment thereof which specifically binds to EGFRvIII and is selected from the group consisting of: a. an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence at least 80% identical to the amino acid sequence of set forth in SEQ ID NO:18 and/or a light chain variable region comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:14; b. an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence at least 80% identical to the amino acid sequence of set forth in SEQ ID NO:10 and/or a light chain variable region comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:6, or c. an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence at least 80% identical to the amino acid sequence of set forth in SEQ ID NO:26 and/or a light chain variable region comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:22. 6.-7. (canceled)
 8. The antigen-binding agent of claim 3, wherein the antigen-binding agent is a chimeric antigen receptor, a bi-specific T-cell engager, a bispecific killer cell engager andor a trispecific killer cell engager and wherein the antigen-binding domain comprises: a. an amino acid sequence at least 80% identical to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:18 and/or an amino acid sequence at least 80% identical to the amino acid sequence of the light chain variable region set forth in SEQ ID NO:14; b. an amino acid sequence at least 80% identical to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:10 and/or an amino acid sequence at least 80% identical to the amino acid sequence of the light chain variable region set forth in SEQ ID NO:6, or c. an amino acid sequence at least 80% identical to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:26 and/or an amino acid sequence at least 80% identical to the amino acid sequence of the light chain variable region set forth in SEQ ID NO:22.
 9. The antigen-binding agent of claim 1, wherein the antigen-binding domain is in the form of a single chain variable fragment (scFv).
 10. The antigen-binding agent of claim 9, wherein the scFv structure is defined by the formula VH-linker-VL.
 11. The antigen-binding agent of claim 8, wherein the chimeric antigen receptor further comprises: a. a transmembrane domain, wherein the antigen-binding domain is optionally connected to the transmembrane domain by a spacer; b. optionally a costimulatory domain, and; c. at least one intracellular signaling domain.
 12. The antigen-binding agent of claim 11, wherein the intracellular signaling domain is selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma R11a, DAP10, and DAP12.
 13. The antigen-binding agent of claim 11 or 12, wherein the chimeric antigen receptor comprises at least one costimulatory domain.
 14. The antigen-binding agent of claim 13, wherein the costimulatory domain is selected from the group consisting of CD28, CD27, 4-1BB, OX40, CD7, B7-1 (CD80), B7-2 (CD86), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D and a combination thereof. 15.-16. (canceled)
 17. The antigen-binding agent of claim 8, wherein the chimeric antigen receptor comprises: a. an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:33; b. an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:34 or as set forth in SEQ ID NO:77; c. an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:30; d. an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:32 or as set forth in SEQ ID NO:74; e. an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:35, or f. an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:36 or as set forth in SEQ ID NO:80. 18.-24. (canceled)
 25. The antigen-binding agent of claim 3, wherein the antigen-binding agent is a bi-specific T-cell engager comprising; a. a CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:15, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:16, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:17, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:19, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:20 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:21, and wherein the bi-specific T-cell engager optionally comprises a CDR3-specific binding domain; b. an amino acid sequence at least 80% identical to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:18 and/or an amino acid sequence at least 80% identical to the amino acid sequence of the light chain variable region set forth in SEQ ID NO:14 and wherein the bi-specific T-cell engager optionally comprises a CDR3-specific binding domain or, c. wherein the bi-specific T-cell engager optionally comprises a CDR3-specific binding domain and an amino acid sequence at least 80% identical to SEQ ID NO:82.
 26. -28. (Cancelled)
 29. An isolated nucleic acid molecule encoding the antigen-binding agent claim
 1. 30. A vector comprising a nucleic acid molecule encoding the antigen-binding agent of claim
 1. 31. (canceled)
 32. An isolated cell comprising or expressing the antigen-binding agent of claim
 1. 33-34. (canceled)
 35. A method of treating subject having a cancer associated with EGFRvIII expression, the method comprising administering the antigen-binding agent of claim
 1. 36.-57. (canceled)
 58. An isolated cell population engineered to express the chimeric antigen receptor of claim
 8. 59. (canceled)
 60. The isolated cell population of claim 58, wherein the isolated cell population comprises immune cells. 61.-64. (canceled)
 65. A pharmaceutical composition comprising the isolated cell population of claim 58 and a pharmaceutically acceptable excipient.
 66. (canceled)
 67. A method of treating a subject having a cancer associated with EGFRvIII expression, the method comprising administering the isolated cell population of claim
 58. 68.-86. (canceled)
 87. The isolated cell population of claim 60, wherein the immune cells comprise T cells, Natural Killer (NK) cells, cytotoxic T cells, regulatory T cells or a combination thereof.
 88. The antigen-binding agent of claim 1, wherein the antigen-binding agent comprises: a. the amino acid sequence of set forth in SEQ ID NO:45; b. an amino acid sequence of at least 80% identical to the amino acid sequence of set forth in SEQ ID NO:45 and comprising a CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:15, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:16, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:17, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:19, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:20 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:21; b. the amino acid sequence of set forth in SEQ ID NO:44; c. an amino acid sequence of at least 80% identical to the amino acid sequence of set forth in SEQ ID NO:44 and comprising a CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:7, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:8, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:9, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:11, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:12 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:13; c. the amino acid sequence of set forth in SEQ ID NO:46; or d. an amino acid sequence of at least 80% identical to the amino acid sequence of set forth in SEQ ID NO:46 and comprising CDRL1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:23, a CDRL2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:24, a CDRL3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:25, a CDRH1 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:27, a CDRH2 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:28 and a CDRH3 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO:29. 